Composition for increasing the half-life of a therapeutic drug in dogs and method of using the same

Abstract

Compositions and methods of use for increasing the half-life of one or more polypeptides in dogs are provided. The compositions comprise a variant canine IgG Fc region.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Patent Application No. 63 / 023,083, filed on 11 May 2020, and U.S. Provisional Patent Application No. 63 / 122,417, filed on 7 December 2020, the contents of which are incorporated herein by reference in their entirety.

[0002] This disclosure generally relates to polypeptides (e.g., fusion polypeptides such as polypeptide-Fc domain fusions; or binding molecules such as ligand-binding moieties of antibody or receptor-Fc fusions) whose half-life in dogs is increased compared to their wild-type equivalents.

[0003] Sequence listings submitted via EFS-WEB The entire contents of the following sequence listing electronically submitted via the USPTO EFS-WEB server are incorporated herein by reference for all purposes, as authorized and provided for in MPEP §1730 II.B.2(a). The sequence listing is contained in the electronically submitted text file identified below:

[0004] File name: 47406-0015WO1_Sequence_Listing.txt

[0005] Date created: May 6, 2021

[0006] Size (bytes): 34,000 bytes [Background technology]

[0007] The Fc region of antibodies plays many, though not limited to, functional roles, including protecting antibodies from degradation via the lysosomal pathway and mediating antibody effector functions. As the use of canine antibodies as therapeutic agents increases, emphasis is being placed not only on selecting the optimal Fab, but also on combining it with the appropriate Fc to achieve the desired half-life and effector function.

[0008] In the art, there are few guidelines regarding increasing the half-life of polypeptide therapeutics (e.g., antibodies) used in dogs. This disclosure addresses this lack by providing Fc region variants that improve the serum persistence of polypeptides (e.g., antibodies) in dogs. [Overview of the project]

[0009] Provided herein are canine Fc (e.g., Fc region variants of canine IgG) or canine FcRn-binding fragments useful for therapeutic polypeptides. This disclosure features polypeptides exhibiting increased binding to canine FcRn than a control polypeptide (e.g., the canine Fc region of wild-type equivalent IgG). In some cases, these polypeptides exhibit increased binding to canine FcRn than the control polypeptide at pH 5.5, pH 6.0, and / or pH 6.5. In some cases, these polypeptides can bind to canine FcRn at higher levels at acidic pH (e.g., pH 5.5, pH 6.0, or pH 6.5) than at neutral pH (e.g., pH 7.0, 7.1, 7.2, 7.3, 7.4, or 7.5). In some cases, these polypeptides bind to canine FcRn at higher levels at pH 5.5 and / or 6.0 than at pH 7.4. This disclosure relates, in part, to polypeptides with increased half-lives in dogs compared to their wild-type equivalents. For example, a binding molecule (e.g., a ligand-binding portion of an antibody or receptor) is provided with an increased half-life compared to a version of the binding molecule not bound to the Fc region or its canine FcRn binding region disclosed herein. Also provided are enzyme-Fc region fusions, ligand-Fc region fusions, nanobody-Fc fusions, and peptide-Fc region fusions, the fusions having increased half-lives compared to their wild-type equivalents. In addition to having one or more substitutions that increase half-life (compared to the wild-type canine Fc region), the Fc region may also include other substitutions that result in, for example, increased effector function, decreased effector function, increased binding to protein A, and / or decreased polypeptide heterogeneity (e.g., by removing one or more post-translational modifications in the Fc region). The canine Fc region sequence may be derived from any canine antibody. In some cases, the canine Fc region sequence is derived from canine IgG (e.g., IgGA, IgGB, IgGC, or IgGD).

[0010] This disclosure comprises a recombinant protein comprising (1) a binding domain or fragment thereof that specifically binds to an epitope of a ligand or protein, wherein the binding domain is (2) bound to a domain comprising an Fc region (CH2+CH3 region) or a canine FcRn binding region as disclosed herein. In some cases, the binding domain comprises (i) six complementarity-determining regions (CDRs) of a canine or human / humanized antibody; (ii) a nanobody; (iii) a soluble receptor-binding domain or ligand-binding fragment thereof that binds to a ligand; and (iv) an extracellular domain of a canine receptor protein.

[0011] This disclosure also provides compositions comprising (1) a first polypeptide comprising a first Fc region (e.g., CH2 region, CH3 region, CH2+CH3 region) comprising a canine IgG Fc region variant described herein, and (2) a second polypeptide comprising a second Fc region comprising a canine IgG Fc region variant described herein. The first and second polypeptides can associate via the first and second Fc regions. In some cases, the amino acid sequences of the first and second Fc regions are the same. In other cases, the amino acid sequences of the first and second Fc regions are different (e.g., amino acids 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25). In some cases, the Fc region variant is a variant of the Fc region of a canine IgGB antibody. In some cases, the Fc region variant is a variant of the Fc region of a canine IgGA antibody. In some cases, the Fc region variant is a variant of the Fc region of a canine IgGC antibody. In some cases, the Fc region variant is a variant of the Fc region of a canine IgGD antibody.

[0012] Fusion molecules comprising Fc region variants and polypeptides of canine IgG disclosed herein are also disclosed. In some cases, the Fc region variant of canine IgG is covalently bound to the polypeptide (e.g., via a hinge region or linker). In some cases, the polypeptide is the ligand-binding domain of a canine receptor protein, the extracellular domain of a canine receptor protein, or the antigen-binding domain. In some cases, the polypeptide is selected from the ligand-binding domain or extracellular domain of canine IL-13Rα1 or IL-13Rα2, canine EPO, canine CTLA4, canine LFA3, canine VEGFR1 / VEGFR3, canine IL-1R, canine GLP-1 receptor agonists, and canine thrombopoietin-binding peptides. In some cases, the polypeptide is an scFv, nanobody, or single-domain antibody. In some cases, the Fc region variant of IgG is a variant of the Fc region of a canine IgGB antibody. In some cases, the Fc region variant of IgG is the same as the Fc region variant of canine IgGA antibody. In some cases, the Fc region variant of IgG is the same as the Fc region variant of canine IgGC antibody. In some cases, the Fc region variant of IgG is the same as the Fc region variant of canine IgGD antibody.

[0013] In some embodiments, the present disclosure relates to polypeptides comprising a canine IgG Fc region variant or its canine FcRn binding region, at a position selected from the group consisting of: (i) The position corresponding to amino acid position 286 of wild-type canine IgG; (ii) The position corresponding to amino acid position 312 of wild-type canine IgG; (iii) the position corresponding to amino acid position 426 of wild-type canine IgG; and (iv) comprising at least one amino acid substitution at the position corresponding to amino acid position 436 of wild-type canine IgG, Here, the amino acid substitution at the position corresponding to amino acid position 286 of wild-type canine IgG is selected from the group consisting of Tyr, Phe, Leu, and Trp, and the amino acid position is based on EU numbering. The polypeptide provides a polypeptide in which the binding affinity to canine FcRn is increased compared to the Fc domain of wild-type canine IgG.

[0014] In some embodiments, at least one amino acid substitution includes an amino acid substitution at a position corresponding to amino acid position 312 of wild-type canine IgG.

[0015] In some embodiments, the polypeptide contains Pro at the amino acid position corresponding to amino acid position 312 of wild-type canine IgG.

[0016] In some embodiments, at least one amino acid substitution includes an amino acid substitution at the position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide includes Tyr, His, or Phe at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide includes Tyr at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide includes His at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide includes Phe at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG.

[0017] In some embodiments, at least one amino acid substitution includes an amino acid substitution at the position corresponding to amino acid position 436 of wild-type canine IgG. In some embodiments, the polypeptide includes His at the amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0018] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80%, 90%, 95%, 98%, 99%, or 100% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9-12.

[0019] In some embodiments, the polypeptide comprises at least one additional amino acid substitution at a position selected from the group consisting of: (i) the amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) the amino acid position corresponding to amino acid position 251 of wild-type canine IgG, (iii) the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iv) the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (v) the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (vi) the amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vii) the amino acid position corresponding to amino acid position 286 of wild-type canine IgG, (viii) the amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (ix) the amino acid position corresponding to amino acid position 308 of wild-type canine IgG, (x) the amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (xi) the amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (xii) the amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (xiii) the amino acid position corresponding to amino acid position 378 of wild-type canine IgG, (xiv) the amino acid position corresponding to amino acid position 380 of wild-type canine IgG, (xv) the amino acid position corresponding to amino acid position 428 of wild-type canine IgG, (xvi) the amino acid position corresponding to amino acid position 430 of wild-type canine IgG, (xvii) The amino acid position corresponding to amino acid position 433 of wild-type canine IgG, (xviii) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and (xix) The amino acid position corresponding to amino acid position 435 of wild-type canine IgG.

[0020] In some embodiments, the polypeptide is (i) Contains Glu or Gln at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) Contains Asp or Glu at the amino acid position corresponding to amino acid position 251 of wild-type canine IgG, (iii) containing Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iv) containing Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (v) Contains Asp, Glu, or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (vi) Contains Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vii) The amino acid position corresponding to amino acid position 286 of wild-type canine IgG contains Asp, Tyr, Phe, Leu, or Trp, (viii) Contains Arg, Gln, or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (ix) Contains Pro at the amino acid position corresponding to amino acid position 308 of wild-type canine IgG, (x) Contains Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (xi) Contains Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (xii) Contains Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (xiii) Contains Val at the amino acid position corresponding to amino acid position 378 of wild-type canine IgG, (xiv) Contains Ala at the amino acid position corresponding to amino acid position 380 of wild-type canine IgG, (xv) Contains Leu at the amino acid position corresponding to amino acid position 428 of wild-type canine IgG, (xvi) Contains Ala or Lys at the amino acid position corresponding to amino acid position 430 of wild-type canine IgG, (xvii) Contains Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG, (xviii) containing Trp, Tyr, Arg, His, Ser, Ala or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and / or (xix) Contains Tyr at the amino acid position corresponding to amino acid position 435 of wild-type canine IgG.

[0021] In some embodiments, at least one amino acid substitution includes an amino acid substitution at the position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide contains Tyr at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide contains Phe at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide contains Leu at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide contains Trp at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0022] In some embodiments, the polypeptide includes at least one additional amino acid substitution at a position selected from the group consisting of: (i) The amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) The amino acid position corresponding to amino acid position 251 of wild-type canine IgG, (iii) The amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iv) The amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (v) The amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (vi) The amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vii) The amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (viii) The amino acid position corresponding to amino acid position 308 of wild-type canine IgG, (ix) The amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (x) The amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (xi) The amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (xii) The amino acid position corresponding to amino acid position 378 of wild-type canine IgG, (xiii) The amino acid position corresponding to amino acid position 380 of wild-type canine IgG, (xiv) The amino acid position corresponding to amino acid position 428 of wild-type canine IgG, (xv) The amino acid position corresponding to amino acid position 430 of wild-type canine IgG, (xvi) The amino acid position corresponding to amino acid position 433 of wild-type canine IgG, (xvii) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and (xviii) The amino acid position corresponding to amino acid position 435 of wild-type canine IgG.

[0023] In some embodiments, the polypeptide is (i) Contains Glu or Gln at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) Contains Asp or Glu at the amino acid position corresponding to amino acid position 251 of wild-type canine IgG, (iii) containing Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iv) containing Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (v) Contains Asp, Glu, or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (vi) Contains Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vii) Contains Arg, Gln, or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (viii) Contains Pro at the amino acid position corresponding to amino acid position 308 of wild-type canine IgG, (ix) Contains Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (x) Contains Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (xi) Contains Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (xii) Contains Val at the amino acid position corresponding to amino acid position 378 of wild-type canine IgG, (xiii) Contains Ala at the amino acid position corresponding to amino acid position 380 of wild-type canine IgG, (xiv) Contains Leu at the amino acid position corresponding to amino acid position 428 of wild-type canine IgG, (xv) Contains Ala or Lys at the amino acid position corresponding to amino acid position 430 of wild-type canine IgG, (xvi) Contains Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG, (xvii) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG contains Trp, Tyr, Arg, His, Ser, Ala or Phe, and / or (xviii) Contains Tyr at the amino acid position corresponding to amino acid position 435 of wild-type canine IgG.

[0024] In some embodiments, at least one additional amino acid substitution is located at a position selected from the group consisting of: (i) The amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) The amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iii) The amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (iv) The amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (v) The amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vi) The amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (vii) The amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (viii) The amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (ix) The amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (x) The amino acid position corresponding to amino acid position 433 of wild-type canine IgG, and (xi) The amino acid position corresponding to amino acid position 434 in wild-type canine IgG.

[0025] In some embodiments, the polypeptide is (i) Contains Glu or Gln at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) containing Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iii) containing Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (iv) containing Asp, Glu, or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (v) Containing Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vi) containing Arg, Gln, or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (vii) Contains Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (viii) Contains Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (ix) Contains Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (x) Contains Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG, and (xi) Contains Trp, Tyr, Arg, His, Ser, Ala, or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0026] In some embodiments, at least one additional amino acid substitution is located at a position selected from the group consisting of: (i) The amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (ii) The amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (iii) The amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and (iv) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0027] In some embodiments, the polypeptide is (i) containing Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (ii) Contains Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (iii) containing Asp, Glu or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and / or (iv) Contains Trp, Tyr, Arg, His, Ser, Ala, or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0028] In some embodiments, the polypeptide is (i) Contains Tyr at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (ii) Contains Thr at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (iii) containing Glu at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and / or (iv) Contains Trp, Tyr, Arg, or His at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0029] In some embodiments, the polypeptide further comprises at least one of the following: (i) Tyr at amino acid position 252, Thr at amino acid position 254, and Glu at amino acid position 256; (ii) Leu at amino acid position 428 and Ser at amino acid position 434; (iii) Asp at amino acid position 256, Arg at amino acid position 307, and Val at amino acid position 311; (iv) Asp at amino acid position 256, Asp at amino acid position 315, and Val at amino acid position 378; (v) Asp at amino acid position 256, Asp, Tyr, Phe, Leu or Trp at amino acid position 286, Arg at amino acid position 307, and Val at amino acid position 311; (vi) Asn at amino acid position 285, Gln at amino acid position 307, and Asp at amino acid position 315; (vii) Asp at amino acid position 256, Arg at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378; (viii) Asp at amino acid position 285, Val at amino acid position 311, and Val at amino acid position 378; (ix) Asp at amino acid position 256, Asp at amino acid position 285, and Val at amino acid position 378; (x) Asp at amino acid position 256, Val at amino acid position 311, and Val at amino acid position 378; (xi) Asp at amino acid position 256, Asp at amino acid position 285, Asp at amino acid position 286, Tyr, Phe, Leu or Trp, Arg at amino acid position 307, and Val at amino acid position 378; (xii) Asp at amino acid position 256, Asp, Tyr, Phe, Leu or Trp at amino acid position 286, Arg at amino acid position 307, Val at amino acid position 311, and Val at position 378; (xiii) Gln at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378; (xiv) Asp at amino acid position 285, Gln at amino acid position 307, and Val at amino acid position 378; (xv) Asp at amino acid position 256, Asp at amino acid position 285, Arg at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378; (xvi) Gln at amino acid position 307, Ala at amino acid position 380, Ser or Ala at amino acid position 434; (xvii) Leu at amino acid position 428, and Ser or Ala at amino acid position 434; or (xviii) Gln at amino acid position 250 and Leu at amino acid position 428.

[0030] In some embodiments, the present disclosure relates to polypeptides comprising a canine IgG Fc region variant or its canine FcRn binding region, comprising two or more positions selected from the group consisting of: (i) The position corresponding to amino acid position 286 of wild-type canine IgG; (ii) The position corresponding to amino acid position 312 of wild-type canine IgG; (iii) The position corresponding to amino acid position 426 of wild-type canine IgG; (iv) the position corresponding to amino acid position 434 of wild-type canine IgG; and (v) Position corresponding to amino acid position 436 of wild-type canine IgG It contains amino acid substitutions, Here, based on EU numbering, the amino acid positions provide a polypeptide with increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG.

[0031] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 286 of wild-type canine IgG is selected from the group consisting of T286L, T286Y, and any of the aforementioned conservative amino acid substitutions.

[0032] The polypeptide according to claim 1, wherein in some embodiments, the amino acid substitution at the position corresponding to amino acid position 312 of wild-type canine IgG is D312P or a conserved amino acid substitution thereof.

[0033] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 426 of wild-type canine IgG is selected from the group consisting of A426Y, A426H, and any of the aforementioned conservative amino acid substitutions.

[0034] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 434 of wild-type canine IgG is N434R or a conserved amino acid substitution thereof.

[0035] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 436 of wild-type canine IgG is Y436H or a conserved amino acid substitution thereof.

[0036] In some embodiments, the polypeptide includes an amino acid substitution at a position corresponding to amino acid position 426 of wild-type canine IgG.

[0037] In some embodiments, the polypeptide includes amino acid substitutions at two or more positions selected from the group consisting of: (i) Positions corresponding to amino acid positions 426 and 286 of wild-type canine IgG; (ii) Positions corresponding to amino acid positions 426 and 312 of wild-type canine IgG; (iii) Positions corresponding to amino acid positions 426 and 434 of wild-type canine IgG; (iv) The positions corresponding to amino acid positions 426 and 436 of wild-type canine IgG; and (v) Positions corresponding to amino acid positions 286, 426, and 436 of wild-type canine IgG.

[0038] In some embodiments, the polypeptide includes amino acid substitutions selected from the group consisting of: (i) A426Y and T286L; (ii) A426Y and D312P; (iii) A426Y and Y436H; (iv) A426H and T286L; (v) A426H and T286Y; (vi) A426H and D312P; and (vii) T286L, A426Y, and Y436H.

[0039] In some embodiments, two or more amino acid substitutions are selected from the group consisting of: (i) A combination of A426Y and one or more of T286L, D312P, N434R, and Y436H; (ii) combinations of A426H with one or more of T286L, T286Y, D312P, N434R and Y436H; and (iii) A combination of N434R and one or more of T286L, T286Y, D312P, and Y436H.

[0040] In some embodiments, wild-type canine IgG is canine IgGA containing an Fc domain having an amino acid sequence identical to at least 80%, at least 85%, at least 90%, or at least 95% of SEQ ID NO: 9; canine IgGB containing an Fc domain having an amino acid sequence identical to at least 80%, at least 85%, at least 90%, or at least 95% of SEQ ID NO: 10; canine IgGC containing an Fc domain having an amino acid sequence identical to at least 80%, at least 85%, at least 90%, or at least 95% of SEQ ID NO: 11; or canine IgGD containing an Fc domain having an amino acid sequence identical to at least 80%, at least 85%, at least 90%, or at least 95% of SEQ ID NO: 12.

[0041] In some embodiments, wild-type canine IgG is canine IgGA, and the Fc region variant of canine IgG or its canine FcRn binding region contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 9. In some embodiments, wild-type canine IgG is canine IgGB, and the Fc region variant of canine IgG or its canine FcRn binding region contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 10. In some embodiments, wild-type canine IgG is canine IgGC, and the Fc region variant of canine IgG or its canine FcRn binding region contains an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 11. In some embodiments, wild-type canine IgG is canine IgGD, and the Fc region variant of canine IgG or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 12.

[0042] In some embodiments, wild-type canine IgG is canine IgGA, and the Fc region variant of canine IgG or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 9.

[0043] In some embodiments, wild-type canine IgG is canine IgGB, and the Fc region variant of canine IgG or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 10.

[0044] In some embodiments, wild-type canine IgG is canine IgGC, and the Fc region variant of canine IgG or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 11.

[0045] In some embodiments, wild-type canine IgG is canine IgGD, and the Fc region variant of canine IgG or its canine FcRn binding region comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, or at least 95% identical to SEQ ID NO: 12.

[0046] In some embodiments, the polypeptide further comprises a binding domain.

[0047] In some embodiments, the binding domain includes (i) six complementarity-determining regions (CDRs) of an immunoglobulin molecule; (ii) a ligand-binding domain of a canine receptor protein; (iii) a nanobody; or (iv) an extracellular domain of a canine receptor protein.

[0048] In some embodiments, the binding domain specifically binds to antigens selected from the group consisting of NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-33, CD3, CD20, CD47, CD52, and the complement system complex.

[0049] In some embodiments, the polypeptide further comprises a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, GLP-1 receptor agonists, and thrombopoietin-binding peptides.

[0050] In some embodiments, the polypeptide binds to canine FcRn at higher levels at acidic pH than at neutral pH in the binding assay. In some embodiments, the polypeptide binds to canine FcRn at higher levels at pH 5.5 than at pH 7.4 in the binding assay. In some embodiments, the polypeptide binds to canine FcRn at higher levels at pH 6.0 than at pH 7.4 in the binding assay.

[0051] In some embodiments, the polypeptide (1) has an increased half-life in dogs compared to one or more control polypeptides, where one or more control polypeptides are identical to one or more polypeptides except that they have the Fc region of the corresponding wild-type canine IgG instead of the Fc region variant of IgG, and / or (2) has increased binding to canine FcRn compared to the control polypeptides, where the amino acid positions are based on EU numbering.

[0052] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 9.

[0053] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 10.

[0054] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 11.

[0055] In some embodiments, the polypeptide comprises an amino acid sequence that is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to SEQ ID NO: 12.

[0056] In some embodiments, the Disclosure provides pharmaceutical compositions comprising (i) a polypeptide described herein and (ii) a pharmaceutically acceptable excipient.

[0057] In some embodiments, this disclosure provides one or more nucleic acids encoding polypeptides described herein.

[0058] In some embodiments, the Disclosure provides one or more expression vectors comprising one or more nucleic acids described herein.

[0059] In some embodiments, the Disclosure provides a host cell comprising one or more nucleic acids or one or more expression vectors as described herein.

[0060] In some embodiments, the Disclosure provides a method for producing one or more polypeptides, the method including: (a) To provide one or more nucleic acids as described herein; (b) Expressing one or more nucleic acids in a host cell culture to produce polypeptides; (c) Recover the polypeptide produced in (b) from the host cell culture.

[0061] In some embodiments, the method further includes formulating a polypeptide as a pharmaceutical formulation.

[0062] In some embodiments, the Disclosure provides a method for treating a disease or disorder in a dog in need thereof, comprising administering to the dog an effective amount of a composition comprising a pharmaceutical composition described herein.

[0063] In some embodiments, the Disclosure provides a method for preventing a disease or disorder in a dog in need thereof, comprising administering to the dog an effective amount of a composition comprising a pharmaceutical composition described herein.

[0064] In some embodiments, the Disclosure provides pharmaceutical compositions described herein for use in a manner that treats a disease or disorder in a dog in need thereof.

[0065] In some embodiments, the Disclosure provides pharmaceutical compositions described herein for use in a manner that prevents diseases or disorders in dogs in need thereof.

[0066] In some embodiments, the disclosure provides the use of polypeptides described herein in the manufacture of a drug for treating a disease or disorder in a dog, in which case it is necessary.

[0067] In some embodiments, the Disclosure provides the use of polypeptides described herein in the manufacture of agents for preventing diseases or disorders in dogs in need thereof.

[0068] In some embodiments, the disease or disorder is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disorder, gastrointestinal disorder, cardiovascular disease, kidney disease, reproductive dysfunction, infection, or cancer.

[0069] In some embodiments, the disease or disorder is atopic dermatitis, allergic dermatitis, osteoarthralgia, arthritis, anemia, or obesity.

[0070] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art in which the present invention pertains. Methods and materials similar to or equivalent to those described herein may be used to implement or verify the present invention, but exemplary methods and materials are described below. All publications, patent applications, patents, and other references referenced herein are incorporated by reference in their entirety. In case of any conflict, including definitions, this application shall prevail. Materials, methods, and examples are illustrative and not intended to limit the scope of the invention.

[0071] Other features and advantages of the present invention will become apparent from the following embodiments and claims for carrying out the invention. [Brief explanation of the drawing]

[0072] [Figure 1] This is the amino acid sequence alignment of canine IgG γ chains. These chains contain VH, CH1, CH2, and CH3 domains, and a hinge region between CH1 and CH2. N-glycosylation sites are shown in bold and enclosed in boxes. These sequences are assigned sequence numbers 13, 14, 15, and 16, respectively. [Figure 2]This is the amino acid sequence alignment of the CH2 region of the canine IgG γ chain. These sequences are assigned sequence numbers 1, 2, 3, and 4, respectively. Substances substituted to increase half-life are underlined. [Figure 3] This is the amino acid sequence alignment of the CH3 region of the canine IgG γ chain. These sequences are assigned the sequence numbers 5, 6, 7, and 8, respectively. Residues substituted to increase half-life are underlined. [Figure 4] This is the amino acid sequence alignment of the Fc region of the canine IgG γ chain. These sequences are assigned sequence numbers 9, 10, 11, and 12, respectively. Residues substituted to increase half-life are underlined. [Figure 5] This table provides EU numbering for the CH2 region of canine IgG. [Figure 6] This table provides EU numbering for the CH3 region of canine IgG. [Figure 7A] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7B] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7C] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7D] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7E]The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7F] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7G] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7H] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7I] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7J] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7K] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7L] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7M] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7N]The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7O] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7P] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7Q] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7R] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7S] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7T] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 7U] The Biacore sensorgrams from alanine scanning mutation introduction experiments are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 8A] The Biacore sensorgrams of different variants of the wild-type and position 250 NNK libraries are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using a 1:1 interaction model. [Figure 8B]The Biacore sensorgrams of different variants of the wild-type and position 250 NNK libraries are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using a 1:1 interaction model. [Figure 8C] The Biacore sensorgrams of different variants of the wild-type and position 250 NNK libraries are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using a 1:1 interaction model. [Figure 9A] The Biacore sensorgrams of different variants of the wild-type and position 252 NNK libraries are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 9B] The Biacore sensorgrams of different variants of the wild-type and position 252 NNK libraries are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 9C] The Biacore sensorgrams of different variants of the wild-type and position 252 NNK libraries are shown. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using the 1:1 interaction model. [Figure 10] A and B show Biacore sensorgrams for wild-type and variant A254T. The thin lines represent measured data, and the thick lines represent approximation curves using a 1:1 interaction model. [Figure 11] A and B show Biacore sensorgrams for wild-type and variant G309P. The thin lines represent measured data, and the thick lines represent approximation curves using a 1:1 interaction model. [Figure 12] A and B show Biacore sensorgrams for wild-type and variant Q311V. The thin lines represent measured data, and the thick lines represent approximation curves using a 1:1 interaction model. [Figure 13]A and B show Biacore sensorgrams for the wild-type and variant D378V. The thin lines represent measured data, and the thick lines represent approximation curves using a 1:1 interaction model. [Figure 14] A and B show Biacore sensorgrams for the wild-type and variant E380A. The thin lines represent measured data, and the thick lines represent the approximation curve using a 1:1 interaction model. [Figure 15A] This shows Biacore sensorgrams of different variants of the wild-type and position 434 NNK libraries. Thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 15B] This shows Biacore sensorgrams of different variants of the wild-type and position 434 NNK libraries. Thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 15C] This shows Biacore sensorgrams of different variants of the wild-type and position 434 NNK libraries. Thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 15D] This shows Biacore sensorgrams of different variants of the wild-type and position 434 NNK libraries. Thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 15E] This shows Biacore sensorgrams of different variants of the wild-type and position 434 NNK libraries. Thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 15F] This shows Biacore sensorgrams of different variants of the wild-type and position 434 NNK libraries. Thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 16]Figures A through E show Biacore sensorgrams of different variants in the concentration series. The canine FcRn concentrations used were 100 nM (white circles), 200 nM (black circles), 400 nM (black triangles), and 800 nM (white triangles). In each figure, thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 17A] The Biacore NNK library sensorgrams of different variants are shown. The concentration of canine FcRn used was 200 nM. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using a 1:1 interaction model. [Figure 17B] The Biacore NNK library sensorgrams of different variants are shown. The concentration of canine FcRn used was 200 nM. In each figure, the thin lines represent the measured data, and the thick lines represent the approximation curve using a 1:1 interaction model. [Figure 18A] The Biacore sensorgrams of different variants in the concentration series are shown. The concentrations of canine FcRn used (from bottom line to top line) were 100 nM, 200 nM, 400 nM, and 800 nM, respectively. Each line is an approximation curve using a 1:1 interaction model. [Figure 18B] The Biacore sensorgrams of different variants in the concentration series are shown. The concentrations of canine FcRn used (from bottom line to top line) were 100 nM, 200 nM, 400 nM, and 800 nM, respectively. Each line is an approximation curve using a 1:1 interaction model. [Figure 19] Figures A-C show Biacore sensorgrams of the binding of wild-type IgGA, IgGA A426Y variant, and IgGA A426H variant to canine FcRn at pH 6.0. The concentrations of canine FcRn used were 50 nM (white square), 100 nM (black circle), 200 nM (white circle), 400 nM (black triangle), and 800 nM (white triangle). In each sensorgram, thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 20]A-F show Biacore sensorgrams of binding of wild-type canine IgGB Fc, single (A426Y), and combined variants of canine IgGB Fc to canine FcRn at pH 6.0. The concentrations of canine FcRn used with wild-type IgG were 200 nM (white circle), 400 nM (black triangle), 800 nM (white triangle), 1600 nM (black diamond), and 3200 nM (white diamond). The concentrations of canine FcRn for the remaining variants were 50 nM (white square), 100 nM (black circle), 200 nM (white circle), 400 nM (black triangle), and 800 nM (white triangle). In each sensorgram, thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 21] A-F show Biacore sensorgrams of the binding of single (A426H) and combined variants of canine IgGB Fc to canine FcRn at pH 6.0. The concentrations of canine FcRn used for the A426H-N434R IgG variant were 200 nM (white circle), 400 nM (black triangle), 800 nM (white triangle), 1600 nM (black diamond), and 3200 nM (white diamond). The concentrations of canine FcRn for the remaining variants were 50 nM (white square), 100 nM (black circle), 200 nM (white circle), 400 nM (black triangle), and 800 nM (white triangle). In each sensorgram, thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 22] Figures A-E show Biacore sensorgrams of the binding of single (N434R) and combined variants of canine IgGB Fc to canine FcRn at pH 6.0. The concentrations of canine FcRn used for the variants were 50 nM (white square), 100 nM (black circle), 200 nM (white circle), 400 nM (black triangle), and 800 nM (white triangle). In each sensorgram, thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 23]This Biacore sensorgram shows the binding of the N434Y variant of canine IgGB Fc to canine FcRn at pH 6.0. The concentrations of canine FcRn used as the variant were 50 nM (white square), 100 nM (black circle), 200 nM (white circle), 400 nM (black triangle), and 800 nM (white triangle). In each sensorgram, the light lines represent the measured data, and the dark lines represent the approximation curve using a 1:1 interaction model. [Figure 24] A-F show Biacore sensorgrams of binding of wild-type canine IgGB Fc, single (A426Y), and combined variants of IgGB Fc to canine FcRn at pH 7.4. In each sensorgram, the lighter lines represent measured data, and the darker lines represent the approximation curve using a 1:1 interaction model. [Figure 25] A-F show Biacore sensorgrams of the binding of single (A426H) and combined variants of IgGB Fc to canine FcRn at pH 7.4. In each sensorgram, the lighter lines represent the measured data, and the darker lines represent the approximation curve using a 1:1 interaction model. [Figure 26] A-E show Biacore sensorgrams of the binding of single (N434R) and combined variants of IgGB Fc to canine FcRn at pH 7.4. In each sensorgram, the lighter lines represent the measured data, and the darker lines represent the approximation curve using a 1:1 interaction model. [Figure 27] This Biacore sensorgram shows the binding of the N434Y variant of IgGB Fc to canine FcRn at pH 7.4. Thin lines represent measured data, and thick lines represent approximation curves using a 1:1 interaction model. [Figure 28] This is the amino acid sequence alignment of canine IgGB Fc and human IgG1 Fc. The positions where amino acid substitutions were made to generate the canine Fc variant (according to EU numbering) are underlined. [Figure 29]This figure shows the terminal phase half-lives (days; Y axis) of canine anti-nerve growth factor (NGF) IgGB Fc variants containing single amino acid substitutions or combinations of amino acid substitutions after intravenous administration to male (M) and female (F) beagles. The animals were randomly divided into eight groups, with males and females in each group. Each animal received a single intravenous dose of 2 mg / kg of antibody, and approximately 1.5 ml of whole blood was collected at the following time points: 0 (before administration), 4 hours after injection, and on days 1, 2, 4, 6, 10, 14, 18, 22, 30, 34, 38, and 42. Serum was separated from the whole blood and assayed for the presence of anti-NGF antibodies by ELISA. Non-compartmental PK analysis (NCA) was performed on each individual serum antibody measurement using PKSolver (Yong Zhang et al., Comput.Methods Programs Biomed.;2010 Sep;99(3):306-14.doi:10.1016 / j.cmpb.2010.01.007). [Figure 30A] This shows Biacore sensorgrams of the binding of different IgGB variants to canine FcRn at pH 5.9. [Figure 30B] This shows Biacore sensorgrams of the binding of different IgGB variants to canine FcRn at pH 5.9. [Figure 30C] This shows Biacore sensorgrams of the binding of different IgGB variants to canine FcRn at pH 5.9. [Figure 30D] This shows Biacore sensorgrams of the binding of different IgGB variants to canine FcRn at pH 5.9. [Figure 30E] This shows Biacore sensorgrams of the binding of different IgGB variants to canine FcRn at pH 5.9. [Figure 30F] This shows Biacore sensorgrams of the binding of different IgGB variants to canine FcRn at pH 5.9. [Figure 30G] This shows Biacore sensorgrams of the binding of different IgGB variants to canine FcRn at pH 5.9. [Figure 30H] This shows Biacore sensorgrams of the binding of different IgGB variants to canine FcRn at pH 5.9. [Figure 30I] This shows Biacore sensorgrams of the binding of different IgGB variants to canine FcRn at pH 5.9. [Figure 30J] This shows Biacore sensorgrams of the binding of different IgGB variants to canine FcRn at pH 5.9. [Figure 30K] This shows Biacore sensorgrams of the binding of different IgGB variants to canine FcRn at pH 5.9. [Figure 31] This is a schematic diagram of a two-compartment pharmacokinetic (PK) model with linear clearance, using a nonlinear mixed-effects model. [Figure 32] A and B show the serum concentrations of different IgGB variants over time. [Figure 33] The predicted serum concentration profiles of antibodies containing wild-type IgGB Fc or IgGB variants A426Y, A426Y+Y436H, A426Y+Y436H+T286L, N434R, N434Y, and YTE are shown. [Figure 34] The structural models of positions 286, 426, and 436 of dog Fc are shown. [Figure 35] The structural models of canine Fc at position A426H are shown. The structure on the left is the FcRn large subunit p51, with the WT structure shown in dark gray and the mutant structure in light gray. The structure on the right is IgGB Fc, with the WT structure shown in dark gray and the mutant structure in light gray. [Figure 36] The structural models of canine Fc at position A426Y are shown. The structure on the left is the FcRn large subunit p51, with the WT structure shown in dark gray and the mutant structure in light gray. The structure on the right is IgGB Fc, with the WT structure shown in dark gray and the mutant structure in light gray. [Figure 37]The structural models of canine Fc at position Y436H are shown. The structure on the left is the FcRn large subunit p51, with the WT structure shown in dark gray and the mutant structure in light gray. The structure on the right is IgGB Fc, with the WT structure shown in dark gray and the mutant structure in light gray. [Figure 38] The structural model of canine Fc at location T286L is shown. The structure on the left (top) is beta-2-microglobulin (WT structure shown in dark gray, mutant structure shown in light gray) and FcRn large subunit p51 (bottom) (WT structure shown in dark gray, mutant structure shown in light gray). The structure on the right is IgGB Fc, with the WT structure shown in dark gray and the mutant structure shown in light gray. [Figure 39] The structural models of canine Fc locations T286L, A426Y, and Y436H are shown. The structure on the left is beta-2-microglobulin and the large FcRn subunit p51. The structure on the right is IgGB Fc. [Modes for carrying out the invention]

[0073] The use of polypeptides (e.g., antibodies, ligand-binding domains of receptors, enzymes, ligands, peptides) is increasing as therapeutic agents for the prevention and treatment of a wide variety of canine diseases. In particular, for the prevention or treatment of chronic diseases that require repeated polypeptide administration, it is important to develop polypeptides with long half-lives.

[0074] Accordingly, this disclosure features canine immunoglobulin Fc regions or canine FcRn binding regions containing mutations that enhance the half-life of one or more polypeptides comprising these sequences. Polypeptides comprising these domains and methods of use thereof are also disclosed. These peptides can be used for a variety of therapeutic and diagnostic applications.

[0075] Where values ​​are given as a range, it should be understood that the description includes disclosure of all possible subranges within that range and specific numerical values ​​that fall within that range, regardless of whether a specific numerical value or subrange is explicitly stated. All numerical notations, such as pH, temperature, time, concentration, and molecular weight, including ranges, are approximations and may vary as appropriate in increments of 1.0 or 0.1 (+) or (-), or by + / - 15%, 10%, 5%, or 2%. It should be understood that all numerical notations are preceded by the word "approximately," although this is not always explicitly stated. It should also be understood that the reagents described herein are merely illustrative, and their equivalents are known in the art, although this is not always explicitly stated.

[0076] When the term "approximately" is used herein to refer to a measurable value such as a quantity or concentration, it means that the specified quantity may vary by 20%, 10%, 5%, 1%, 0.5%, or 0.1%.

[0077] Canine antibodies Dogs have four IgG heavy chains called A, B, C, and D. These heavy chains represent four different subclasses of canine IgG, called IgGA, IgGB, IgGC, and IgGD. The amino acid and DNA sequences of these heavy chains are available from Tang et al., Vet. Immunol. Immunopathol., 80:259-270 (2001) and the GENBANK database. For example, the amino acid sequence of the IgGA heavy chain is GENBANK accession number AAL35301.1, IgGB is GENBANK accession number AAL35302.1, IgGC is GENBANK accession number AAL35303.1, and IgGD is GENBANK accession number AAL35304.1. Canine antibodies also contain two types of light chains: kappa and lambda. The DNA and amino acid sequences of these light chains are also available from the GENBANK database. For example, the amino acid sequence of the canine kappa light chain has accession number ABY57289.1, and the canine lambda light chain has accession number ABY55569.1.

[0078] CH2 region of the canine Fc region: The CH2 region of a canine antibody contains or consists of amino acids 237–340 (according to EU numbering) of a canine IgG antibody. It is understood that the CH2 region may contain 1–6 (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at its N-terminus and / or C-terminus.

[0079] The amino acid sequence of the CH2 region of canine IgGA is provided below: GPSVLI FPPKPKDILR ITRTPEVTCV VLDLGREDPE VQISWFVDGK EVHTAKTQSR EQQFNGTYRV VSVLPIEHQD WLTGKEFKCR VNHIDLPSPI ERTISKAR(Sequence ID 1)

[0080] The amino acid sequence of the CH2 domain of canine IgGB is provided below: GPSVFIFPPK PKDTLLIART PEVTCVVVDL DPEDPEVQIS WFVDGKQMQT AKTQPREEQF NGTYRVVSVL PIGHQDWLKG KQFTCKVNNK ALPSPIERTI SKAR (Sequence ID 2)

[0081] The amino acid sequence of the CH2 domain of canine IgGC is provided below: GPSVFIFPP KPKDILVTAR TPTVTCVVVD LDPENPEVQI SWFVDSKQVQ TANTQPREEQ SNGTYRVVSV LPIGHQDWLS GKQFKCKVNN KALPSPIEEI ISKTP(Sequence ID 3)

[0082] The amino acid sequence of the CH2 domain of canine IgGD is provided below: GPSV FIFPPKPKDI LRITRTPEIT CVVLDLGRED PEVQISWFVD GKEVHTAKTQ PREQQFNSTY RVVSVLPIEH QDWLTGKEFK CRVNHIGLPS PIERTISKAR(Sequence ID 4)

[0083] CH3 region of the canine Fc domain: The CH3 region of a canine antibody contains or consists of amino acids 345–447 (according to EU numbering) of a canine IgG antibody. It is understood that the CH3 region may contain 1–6 (e.g., 1, 2, 3, 4, 5, 6) additional amino acids or deletions at its N-terminus and / or C-terminus.

[0084] The amino acid sequence of the CH3 domain of canine IgGA is provided below: KPSVYVLP PSPKELSSSD TVSITCLIKD FYPPDIDVEW QSNGQQEPER KHRMTPPQLD EDGSYFLYSK LSVDKSRWQQ GDPFTCAVMH ETLQNHYTDL SLSHSPGK(Sequence ID 5)

[0085] The amino acid sequence of the CH3 domain in canine IgGB is provided below: QP SVYVLPPSRE ELSKNTVSLT CLIKDFFPPD IDVEWQSNGQ QEPESKYRTT PPQLDEDGSY FLYSKLSVDK SRWQRGDTFI CAVMHEALHN HYTQESLSHS PGK(Sequence ID 6)

[0086] The amino acid sequence of the CH3 domain of canine IgGC is provided below: Q PNVYVLPPSR DEMSKNTVTL TCLVKDFFPP EIDVEWQSNG QQEPESKYRM TPPQLDEDGS YFLYSKLSVD KSRWQRGDTF ICAVMHEALH NHYTQISLSH SPGK(Sequence ID 7)

[0087] The amino acid sequence of the CH3 domain of canine IgGD is provided below: QPSVYV LPPSPKELSS SDTVTLTCLI KDFFPPEIDV EWQSNGQPEP ESKYHTTAPQ LDEDGSYFLY SKLSVDKSRW QQGDTFTCAV MHEALQNHYT DLSLSHSPGK(Sequence ID 8)

[0088] Fc region of the canine Fc region: The Fc region of the canine IgG antibody contains or consists of amino acids 231-447 (according to EU numbering).

[0089] The amino acid sequence of the Fc domain of canine IgGA is provided below: VPEPLGGPSVLI FPPKPKDILR ITRTPEVTCV VLDLGREDPE VQISWFVDGK EVHTAKTQSR EQQFNGTYRV VSVLPIEHQD WLTGKEFKCR VNHIDLPSPI ERTISKARGR AHKPSVYVLP PSPKELSSSD TVSITCLIKD FYPPDIDVEW QSNGQQEPER KHRMTPPQLD EDGSYFLYSK LSVDKSRWQQ GDPFTCAVMH ETLQNHYTDL SLSHSPGK (Sequence ID 9)

[0090] The amino acid sequence of the Fc domain of canine IgGB is provided below: APEMLGGPSVFIFPPK PKDTLLIART PEVTCVVVDL DPEDPEVQIS WFVDGKQMQT AKTQPREEQF NGTYRVVSVL PIGHQDWLKG KQFTCKVNNK ALPSPIERTI SKARGQAHQP SVYVLPPSRE ELSKNTVSLT CLIKDFFPPD IDVEWQSNGQ QEPESKYRTT PPQLDEDGSY FLYSKLSVDK SRWQRGDTFI CAVMHEALHN HYTQESLSHS PGK (Sequence ID 10)

[0091] The amino acid sequence of the Fc domain of canine IgGC is provided below: GCGLLGGPSVFIFPP KPKDILVTAR TPTVTCVVVD LDPENPEVQI SWFVDSKQVQ TANTQPREEQ SNGTYRVVSV LPIGHQDWLS GKQFKCKVNN KALPSPIEEI ISKTPGQAHQ PNVYVLPPSR DEMSKNTVTL TCLVKDFFPP EIDVEWQSNG QQEPESKYRM TPPQLDEDGS YFLYSKLSVD KSRWQRGDTF ICAVMHEALH NHYTQISLSH SPGK(Sequence ID 11)

[0092] The amino acid sequence of the Fc domain of canine IgGD is provided below: VPESLGGPSV FIFPPKPKDI LRITRTPEIT CVVLDLGRED PEVQISWFVD GKEVHTAKTQ PREQQFNSTY RVVSVLPIEH QDWLTGKEFK CRVNHIGLPS PIERTISKAR GQAHQPSVYV LPPSPKELSS SDTVTLTCLI KDFFPPEIDV EWQSNGQPEP ESKYHTTAPQ LDEDGSYFLY SKLSVDKSRW QQGDTFTCAV MHEALQNHYT DLSLSHSPGK(Sequence ID 12)

[0093] Substitution of canine IgG Fc to improve half-life Increased serum persistence is a beneficial property for therapeutic polypeptides. This disclosure features substitutions of the Fc region of wild-type canine IgGA, IgGB, IgGC, and IgGD, the substitutions improving the half-life in dogs of one or more polypeptides containing these Fc regions compared to one or more control polypeptides, where one or more control polypeptides are identical to one or more polypeptides except that they have the Fc region of the corresponding wild-type canine IgG instead of the Fc region variant of IgG. Substitutions that increase half-life may be made in one or more of the canine CH2 region, canine CH3 region, or in association with the canine Fc (e.g., CH2+CH3) region.

[0094] This disclosure relates to a polypeptide comprising a canine IgG Fc domain or a canine FcRn binding region, at a position selected from the group consisting of: (i) The position corresponding to amino acid position 286 of wild-type canine IgG; (ii) The position corresponding to amino acid position 312 of wild-type canine IgG; (iii) the position corresponding to amino acid position 426 of wild-type canine IgG; and (iv) Position corresponding to amino acid position 436 of wild-type canine IgG It contains at least one amino acid substitution, Here, the amino acid substitution at the position corresponding to amino acid position 286 of wild-type canine IgG is selected from the group consisting of Tyr, Phe, Leu, and Trp, and the amino acid position is based on EU numbering, providing a polypeptide that has increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG. In some embodiments, the polypeptide has increased binding affinity to canine FcRn at a pH of about 5.0 to about 6.5 (e.g., about 5.5 or about 6.0) compared to the Fc domain of wild-type canine IgG in a binding assay. In some embodiments, the binding assay refers to a comparative assay in which the binding affinity of the polypeptide variant described herein to canine FcRn at a pH of, for example, about 5.0 to about 6.5 is compared to the binding affinity of wild-type canine IgG to canine FcRn at the same pH (e.g., pH of about 5.0 to about 6.5). In some embodiments, the binding assay is carried out using equivalent conditions. In some embodiments, the binding assay is a surface plasmon resonance (SPR) assay.

[0095] In some embodiments, at least one amino acid substitution includes an amino acid substitution at a position corresponding to amino acid position 312 of wild-type canine IgG.

[0096] In some embodiments, the polypeptide contains Pro at the amino acid position corresponding to amino acid position 312 of wild-type canine IgG.

[0097] In some embodiments, at least one amino acid substitution includes an amino acid substitution at the position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide includes Tyr, His, or Phe at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide includes Tyr at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide includes His at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG. In some embodiments, the polypeptide includes Phe at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG.

[0098] In some embodiments, at least one amino acid substitution includes an amino acid substitution at the position corresponding to amino acid position 436 of wild-type canine IgG. In some embodiments, the polypeptide includes His at the amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0099] In some embodiments, the polypeptide contains an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 12.

[0100] In some cases, the Disclosure provides CH2 region variants of canine IgG that include an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs: 1-4. The Disclosure also provides CH2 region variants of canine IgG that include an amino acid sequence which differs by 1 to 15 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) from any one of SEQ ID NOs: 1-4.

[0101] In other cases, the Disclosure features CH3 region variants of canine IgG containing amino acid sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the amino acid sequences described in SEQ ID NOs. The Disclosure also features CH3 region variants of canine IgG containing amino acid sequences that differ by 1 to 15 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) from any one of SEQ ID NOs.

[0102] In certain cases, the Disclosure features Fc region variants of canine IgG that include an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs. The Disclosure also discloses Fc region variants of canine IgG that include an amino acid sequence which differs from any one of SEQ ID NOs. by 1 to 20 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids).

[0103] In some cases, at least one of the following regions (e.g., 1, 2, or 3) in the Fc CH2 region variant of canine IgG is identical to the corresponding region in the Fc CH2 region of wild-type canine IgG: Amino acid positions 250-256; Amino acid positions 285-288; and Amino acid positions 307-315 (Here, amino acid positions are based on EU numbering.) In some cases, all of the above regions in the Fc CH2 region variant of canine IgG are identical to the corresponding regions in the Fc CH2 region of wild-type canine IgG.

[0104] In some cases, at least one (e.g., one or two) of the following regions in the Fc CH3 region variant of canine IgG is identical to the corresponding region in the Fc CH3 region of wild-type canine IgG: Amino acid positions 376-380; and Amino acid positions 428-436 (Here, amino acid positions are based on EU numbering.) In some cases, all of the above regions in the Fc CH3 region variant of canine IgG are identical to the corresponding regions in the Fc CH3 region of wild-type canine IgG.

[0105] In some cases, at least one of the following regions (e.g., 1, 2, 3, 4, or 5) in the Fc variant of canine IgG is identical to the corresponding region in the Fc of wild-type canine IgG: Amino acid positions 250-256; Amino acid positions 285-288; Amino acid positions 307-315; Amino acid positions 376-380; and Amino acid positions 428-436 (Here, amino acid positions are based on EU numbering.) In some cases, all of the following regions in the Fc variant of canine IgG are identical to the corresponding regions in the Fc of wild-type canine IgG.

[0106] In some cases, at least one of the following regions (e.g., 1, 2, 3, 4, or 5) in the Fc variant of canine IgG is identical to the corresponding region in the Fc of wild-type canine IgG: Amino acid positions 250-256; Amino acid positions 285, 287, and 288; Amino acid positions 307-315; Amino acid positions 376-380; and Amino acid positions 428-436 (Here, amino acid positions are based on EU numbering.) In some cases, all of the following regions in the Fc variant of canine IgG are identical to the corresponding regions in the Fc of wild-type canine IgG.

[0107] In some embodiments, one or more polypeptides comprising a canine IgG Fc CH2 region variant are provided, wherein the CH2 region variant comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs: 1 to 4.

[0108] In some embodiments, the polypeptides are characterized by one or more polypeptides comprising a canine IgG Fc CH3 region variant, wherein the CH3 region variant comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs. 5 to 8.

[0109] In some embodiments, the polypeptide comprises one or more polypeptides containing an Fc region variant of canine IgG, wherein the Fc region variant comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs: 9 to 12.

[0110] In some embodiments, the polypeptide is (i) containing Pro at the amino acid position corresponding to amino acid position 312 of wild-type canine IgG, and / or (ii) containing Tyr, His or Phe at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG, and / or (iii) Contains Tyr, Phe, Leu, and Trp at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0111] In some embodiments, the polypeptide is (i) containing Pro at the amino acid position corresponding to amino acid position 312 of wild-type canine IgG, and / or (ii) containing Tyr at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG, and / or (iii) Contains Tyr, Phe, Leu, and Trp at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0112] In some embodiments, the polypeptide is (i) containing Pro at the amino acid position corresponding to amino acid position 312 of wild-type canine IgG, and / or (ii) containing His at the amino acid position corresponding to amino acid position 426 of wild-type canine IgG, and / or (iii) Contains Tyr, Phe, Leu, and Trp at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG.

[0113] As noted elsewhere, in some embodiments, the polypeptide further includes at least one additional amino acid substitution in the region corresponding to amino acid positions 250-256, 285-288, 307-315, 376-380, or 428-436 of wild-type canine IgG, where the amino acid positions are based on EU numbering, and the polypeptide exhibits increased binding to canine FcRn compared to the Fc domain of wild-type canine IgG.

[0114] In some embodiments, the polypeptide further comprises at least one additional amino acid substitution in the region corresponding to amino acid positions 250–256, 285, 287 and 288; amino acid positions 307–315; amino acid positions 376–380; or amino acid positions 428–436 of wild-type canine IgG, where the amino acid positions are based on EU numbering, and the polypeptide exhibits increased binding to canine FcRn compared to the Fc domain of wild-type canine IgG. The at least one additional amino acid substitution encompassed by this disclosure comprises one or more of the amino acid substitutions disclosed in Table 1 (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20). [Table 1]

[0115] In some cases, at least one additional amino acid substitution encompassed by this disclosure includes one or more substitutions disclosed in Table 2 (e.g., 1, 2, 3, or 4). [Table 2]

[0116] All possible combinations and permutations of the substitutions disclosed above are incorporated herein. In some cases, the polypeptide comprises at least one additional amino acid substitution (e.g., two or more, three or more, four or more, five or more) selected from the group consisting of: (i) Tyr at amino acid position 252, Thr at amino acid position 254, and Glu at amino acid position 256; (ii) Leu at amino acid position 428 and Ser at amino acid position 434; (iii) Asp at amino acid position 256, Arg at amino acid position 307, and Val at amino acid position 311; (iv) Asp at amino acid position 256, Asp at amino acid position 315, and Val at amino acid position 378; (v) Asp at amino acid position 256, Asp, Tyr, Phe, Leu or Trp at amino acid position 286, Arg at amino acid position 307, and Val at amino acid position 311; (vi) Asn at amino acid position 285, Gln at amino acid position 307, and Asp at amino acid position 315; (vii) Asp at amino acid position 256, Arg at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378; (viii) Asp at amino acid position 285, Val at amino acid position 311, and Val at amino acid position 378; (ix) Asp at amino acid position 256, Asp at amino acid position 285, and Val at amino acid position 378; (x) Asp at amino acid position 256, Val at amino acid position 311, and Val at amino acid position 378; (xi) Asp at amino acid position 256, Asp at amino acid position 285, Asp at amino acid position 286, Tyr, Phe, Leu or Trp, Arg at amino acid position 307, and Val at amino acid position 378; (xii) Asp at amino acid position 256, Asp at amino acid position 286, Arg at amino acid position 307, Val at amino acid position 311, and Val at position 378; (xiii) Gln at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378; (xiv) Asp at amino acid position 285, Gln at amino acid position 307, and Val at amino acid position 378; (xv) Asp at amino acid position 256, Asp at amino acid position 285, Arg at amino acid position 307, Val at amino acid position 311, and Val at amino acid position 378; (xvi) Gln at amino acid position 307, Ala at amino acid position 380, Ser or Ala at amino acid position 434; (xvii) Leu at amino acid position 428, and Ser or Ala at amino acid position 434; (xviii) Gln at amino acid position 250 and Leu at amino acid position 428; (xix) Glu at amino acid position 250 and Glu at amino acid position 251; (xx) Phe at amino acid position 256 and Phe at amino acid position 309; (xxi) Ala at amino acid position 430 and Lys at amino acid position 433; (xxii) Phe at amino acid position 434 and His at amino acid position 436; and (xxiii) Tyr at amino acid position 435 and His at amino acid position 436.

[0117] In some cases, the substitution does not involve the combination of Tyr at amino acid position 252, Thr at amino acid position 254, and Glu at amino acid position 256.

[0118] In some embodiments, at least one additional amino acid substitution is located at a position selected from the group consisting of: (i) The amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) The amino acid position corresponding to amino acid position 251 of wild-type canine IgG, (iii) The amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iv) The amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (v) The amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (vi) The amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vii) The amino acid position corresponding to amino acid position 286 of wild-type canine IgG, (viii) The amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (ix) The amino acid position corresponding to amino acid position 308 of wild-type canine IgG, (x) The amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (xi) The amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (xii) The amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (xiii) The amino acid position corresponding to amino acid position 378 of wild-type canine IgG, (xiv) The amino acid position corresponding to amino acid position 380 of wild-type canine IgG, (xv) The amino acid position corresponding to amino acid position 428 of wild-type canine IgG, (xvi) The amino acid position corresponding to amino acid position 430 of wild-type canine IgG, (xvii) The amino acid position corresponding to amino acid position 433 of wild-type canine IgG, (xviii) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG, (xix) The amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and (xx) The amino acid position corresponding to amino acid position 436 in wild-type canine IgG.

[0119] In some embodiments, the polypeptide is (i) Contains Glu or Gln at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) Contains Asp or Glu at the amino acid position corresponding to amino acid position 251 of wild-type canine IgG, (iii) containing Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iv) containing Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (v) Contains Asp, Glu, or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (vi) Contains Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vii) The amino acid position corresponding to amino acid position 286 of wild-type canine IgG contains Asp, Tyr, Phe, Leu, or Trp, (viii) Contains Arg, Gln, or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (ix) Contains Pro at the amino acid position corresponding to amino acid position 308 of wild-type canine IgG, (x) Contains Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (xi) Contains Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (xii) Contains Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (xiii) Contains Val at the amino acid position corresponding to amino acid position 378 of wild-type canine IgG, (xiv) Contains Ala at the amino acid position corresponding to amino acid position 380 of wild-type canine IgG, (xv) Contains Leu at the amino acid position corresponding to amino acid position 428 of wild-type canine IgG, (xvi) Contains Ala or Lys at the amino acid position corresponding to amino acid position 430 of wild-type canine IgG, (xvii) Contains Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG, (xviii) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG contains Trp, Tyr, Arg, His, Ser, Ala or Phe, (xix) Contains Tyr at the amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and / or (xx) Contains His at the amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0120] In some embodiments, at least one amino acid substitution includes an amino acid substitution at the position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide includes Tyr at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide includes Phe at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide includes Leu at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide includes Trp at the amino acid position corresponding to amino acid position 286 of wild-type canine IgG. In some embodiments, the polypeptide includes at least one additional amino acid substitution at a position selected from the group consisting of: (i) The amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) The amino acid position corresponding to amino acid position 251 of wild-type canine IgG, (iii) The amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iv) The amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (v) The amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (vi) The amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vii) The amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (viii) The amino acid position corresponding to amino acid position 308 of wild-type canine IgG, (ix) The amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (x) The amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (xi) The amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (xii) The amino acid position corresponding to amino acid position 378 of wild-type canine IgG, (xiii) The amino acid position corresponding to amino acid position 380 of wild-type canine IgG, (xiv) The amino acid position corresponding to amino acid position 428 of wild-type canine IgG, (xv) The amino acid position corresponding to amino acid position 430 of wild-type canine IgG, (xvi) The amino acid position corresponding to amino acid position 433 of wild-type canine IgG, (xvii) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG, (xviii) The amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and (xix) The amino acid position corresponding to amino acid position 436 in wild-type canine IgG.

[0121] In some embodiments, the polypeptide is (i) Contains Glu or Gln at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) Contains Asp or Glu at the amino acid position corresponding to amino acid position 251 of wild-type canine IgG, (iii) containing Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iv) containing Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (v) Contains Asp, Glu, or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (vi) Contains Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vii) Contains Arg, Gln, or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (viii) Contains Pro at the amino acid position corresponding to amino acid position 308 of wild-type canine IgG, (ix) Contains Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (x) Contains Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (xi) Contains Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (xii) Contains Val at the amino acid position corresponding to amino acid position 378 of wild-type canine IgG, (xiii) Contains Ala at the amino acid position corresponding to amino acid position 380 of wild-type canine IgG, (xiv) Contains Leu at the amino acid position corresponding to amino acid position 428 of wild-type canine IgG, (xv) Contains Ala or Lys at the amino acid position corresponding to amino acid position 430 of wild-type canine IgG, (xvi) Contains Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG, (xvii) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG contains Trp, Tyr, Arg, His, Ser, Ala, or Phe, (xviii) containing Tyr at the amino acid position corresponding to amino acid position 435 of wild-type canine IgG, and / or (xix) Contains His at the amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0122] In some embodiments, at least one additional amino acid substitution is located at a position selected from the group consisting of: (i) The amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) The amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iii) The amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (iv) The amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (v) The amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vi) The amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (vii) The amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (viii) The amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (ix) The amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (x) The amino acid position corresponding to amino acid position 433 of wild-type canine IgG, (xi) The amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and (xii) The amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0123] In some embodiments, the polypeptide (i) contains Glu or Gln at the amino acid position corresponding to amino acid position 250 of wild-type canine IgG, (ii) contains Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (iii) contains Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (iv) contains Asp, Glu or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, (v) contains Asn or Asp at the amino acid position corresponding to amino acid position 285 of wild-type canine IgG, (vi) contains Arg, Gln or Ala at the amino acid position corresponding to amino acid position 307 of wild-type canine IgG, (vii) contains Pro at the amino acid position corresponding to amino acid position 309 of wild-type canine IgG, (viii) contains Val at the amino acid position corresponding to amino acid position 311 of wild-type canine IgG, (ix) contains Asp at the amino acid position corresponding to amino acid position 315 of wild-type canine IgG, (x) contains Lys at the amino acid position corresponding to amino acid position 433 of wild-type canine IgG, (xi) contains Trp, Tyr, Arg, His, Ser, Ala or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG, and (xii) contains His at the amino acid position corresponding to amino acid position 436 of wild-type canine IgG.

[0124] In some embodiments, at least one additional amino acid substitution is at a position selected from the group consisting of: (i) the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (ii) the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (iii) the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and (iv) the amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0125] In some embodiments, the polypeptide (i) contains Tyr or Met at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (ii) contains Thr or Ser at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (iii) contains Asp, Glu or Phe at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and / or (iv) contains Trp, Tyr, Arg, His, Ser, Ala or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0126] ]> In some embodiments, the polypeptide (i) contains Tyr at the amino acid position corresponding to amino acid position 252 of wild-type canine IgG, (ii) contains Thr at the amino acid position corresponding to amino acid position 254 of wild-type canine IgG, (iii) contains Glu at the amino acid position corresponding to amino acid position 256 of wild-type canine IgG, and / or (iv) contains Trp, Tyr, Arg or His at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG.

[0127] Substitutions may be made on one or both strands of the CH2 domain, CH3 domain, or Fc domain. In some cases, substitutions on both strands of the CH2 domain, CH3 domain, or Fc domain are identical. In some cases, substitutions on both strands of the CH2 domain, CH3 domain, or Fc domain are not identical. In some cases, the Fc region contains one or more additional substitutions that result in an increase or decrease in effector function and / or improvement of product heterogeneity.

[0128] This disclosure also relates to polypeptides comprising a canine IgG Fc region variant or its canine FcRn binding region, wherein two or more positions (e.g., 2, 3, 4, or 5) are selected from the group consisting of: (i) The position corresponding to amino acid position 286 of wild-type canine IgG; (ii) The position corresponding to amino acid position 312 of wild-type canine IgG; (iii) The position corresponding to amino acid position 426 of wild-type canine IgG; (iv) the position corresponding to amino acid position 434 of wild-type canine IgG; and (v) Position corresponding to amino acid position 436 of wild-type canine IgG It contains amino acid substitutions, Here, based on EU numbering, the amino acid positions provide a polypeptide with increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG.

[0129] All possible combinations and permutations of substitutions disclosed herein are incorporated herein. In some embodiments, two or more amino acid substitutions produce a synergistic effect insofar as they increase the binding affinity of the polypeptide to canine FcRn compared to a polypeptide containing only one of the two or more amino acid substitutions described herein.

[0130] In some embodiments, the polypeptides described herein include at least one additional amino acid substitution at a position other than those corresponding to positions 286, 312, 426, 434, and 436 of wild-type canine IgG. For example, the polypeptides described herein may include about 1, 2, 3, 4, or 5 to about 30 additional amino acid substitutions of canine IgG.

[0131] In some embodiments, polypeptides exhibit increased binding affinity to canine FcRn at pH levels of approximately 5.0 to 6.5 (e.g., approximately 5.5 or 6.0) compared to the Fc domain of wild-type canine IgG. Methods for determining FcRn binding affinity are well known to those skilled in the art, and examples thereof are described separately herein.

[0132] Differences in FcRn binding activity can be suitably determined using equivalent or similar assays for each variable (e.g., pH value, number of amino acid substitutions, position of amino acid substitutions, type of amino acid substitutions, etc.). In this context, equivalent assays refer to assays performed in substantially the same or similar manner to minimize or avoid unnecessary variables that could substantially affect the results, independent of the method by which the assay is performed and the variable being evaluated. However, it is understood that the conditions required to perform an assay determining FcRn binding at pH 6.0 may differ from the conditions required to perform a similar assay at pH 7.4, for example, considering the effect of pH on the method by which the assay is performed. In some embodiments, polypeptides exhibit increased binding affinity to canine FcRn at pH approximately 5.0 to approximately 6.5 (e.g., approximately 5.5 or approximately 6.0) when compared to the Fc domain of wild-type canine IgG using equivalent assays.

[0133] In some embodiments, the polypeptide binds to canine FcRn at a higher level at acidic pH than at neutral pH in an equivalent assay. In some embodiments, the polypeptide binds to canine FcRn at a higher level at pH 5.5 than at pH 7.4 in an equivalent assay. In some embodiments, the polypeptide binds to canine FcRn at a higher level at pH 6.0 than at pH 7.4 in an equivalent assay.

[0134] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 286 of wild-type canine IgG is selected from the group consisting of T286L, T286Y, and any of the aforementioned conservative amino acid substitutions.

[0135] The polypeptide according to claim 1, wherein in some embodiments, the amino acid substitution at the position corresponding to amino acid position 312 of wild-type canine IgG is D312P or its conservative amino acid substitution.

[0136] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 426 of wild-type canine IgG is selected from the group consisting of A426Y, A426H, and any of the aforementioned conservative amino acid substitutions.

[0137] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 434 of wild-type canine IgG is N434R or its conservative amino acid substitution.

[0138] In some embodiments, the amino acid substitution at the position corresponding to amino acid position 436 of wild-type canine IgG is Y436H or its conservative amino acid substitution.

[0139] In some embodiments, the polypeptide comprises an amino acid substitution at the position corresponding to amino acid position 426 of wild-type canine IgG.

[0140] As used herein, “conservative amino acid substitution” refers to the substitution of one amino acid residue with another amino acid residue that has similar properties, such as charge, hydrophobicity, and size. In some embodiments, a conservative amino acid substitution refers to a substitution that results in similar properties or functions to another amino acid substitution. For example, the conservative amino acid substitution of A426Y may be A426F or A426T.

[0141] In some embodiments, the polypeptide includes amino acid substitutions at two or more positions selected from the group consisting of: (i) Positions corresponding to amino acid positions 426 and 286 of wild-type canine IgG; (ii) Positions corresponding to amino acid positions 426 and 312 of wild-type canine IgG; (iii) the positions corresponding to amino acid positions 426 and 434 of wild-type canine IgG; and (iv) Positions corresponding to amino acid positions 426 and 436 of wild-type canine IgG.

[0142] In some embodiments, two or more amino acid substitutions are selected from the group consisting of: (i) A combination of A426Y and one or more of T286L, T286Y, D312P, N434R, and Y436H; (ii) combinations of A426H with one or more of T286L, T286Y, D312P, N434R and Y436H; and (iii) A combination of N434R and one or more of T286L, T286Y, D312P, and Y436H.

[0143] In some embodiments, the polypeptide includes amino acid substitutions selected from the group consisting of: (i) A426Y and T286L; (ii) A426Y and D312P; (iii) A426Y and Y436H; (iv) A426H and T286L; (v) A426H and T286Y; and (vi) A426H and D312P.

[0144] In some embodiments, the polypeptide contains an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 12.

[0145] In some cases, the Disclosure provides CH2 region variants of canine IgG that include an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs: 1-4. The Disclosure also provides CH2 region variants of canine IgG that include an amino acid sequence which differs by 1 to 15 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) from any one of SEQ ID NOs: 1-4.

[0146] In other cases, the Disclosure features CH3 region variants of canine IgG containing amino acid sequences that are at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to any one of the amino acid sequences described in SEQ ID NOs. The Disclosure also features CH3 region variants of canine IgG containing amino acid sequences that differ by 1 to 15 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15) from any one of SEQ ID NOs.

[0147] In certain cases, the Disclosure features Fc region variants of canine IgG that include an amino acid sequence which is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs. The Disclosure also discloses Fc region variants of canine IgG that include an amino acid sequence which differs from any one of SEQ ID NOs. by 1 to 20 amino acids (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 amino acids).

[0148] In some cases, at least one of the following regions (e.g., 1, 2, or 3) in the Fc CH2 region variant of canine IgG is identical to the corresponding region in the Fc CH2 region of wild-type canine IgG: Amino acid positions 250-256; and Amino acid positions 307-311 (Here, amino acid positions are based on EU numbering.) In some cases, all of the above regions in the Fc CH2 region variant of canine IgG are identical to the corresponding regions in the Fc CH2 region of wild-type canine IgG.

[0149] In some cases, at least one (e.g., one or two) of the following regions in the Fc CH3 region variant of canine IgG is identical to the corresponding region in the Fc CH3 region of wild-type canine IgG: Amino acid positions 376-380; and Amino acid positions 428-433 (Here, amino acid positions are based on EU numbering.) In some cases, all of the above regions in the Fc CH3 region variant of canine IgG are identical to the corresponding regions in the Fc CH3 region of wild-type canine IgG.

[0150] In some cases, at least one of the following regions (e.g., 1, 2, 3, 4, or 5) in the Fc variant of canine IgG is identical to the corresponding region in the Fc of wild-type canine IgG: Amino acid positions 250-256; Amino acid positions 307-311; Amino acid positions 376-380; and Amino acid positions 428-433 (Here, amino acid positions are based on EU numbering.) In some cases, all of the following regions in the Fc variant of canine IgG are identical to the corresponding regions in the Fc of wild-type canine IgG.

[0151] In some cases, at least one of the following regions (e.g., 1, 2, 3, 4, or 5) in the Fc variant of canine IgG is identical to the corresponding region in the Fc of wild-type canine IgG: Amino acid positions 250-256; Amino acid positions 285, 287, and 288; Amino acid positions 307-311; Amino acid positions 376-380; and Amino acid positions 428-433 (Here, amino acid positions are based on EU numbering.) In some cases, all of the following regions in the Fc variant of canine IgG are identical to the corresponding regions in the Fc of wild-type canine IgG.

[0152] In some embodiments, one or more polypeptides comprising a canine IgG Fc CH2 region variant are provided, wherein the CH2 region variant comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs: 1 to 4.

[0153] In some embodiments, the polypeptides are characterized by one or more polypeptides comprising a canine IgG Fc CH3 region variant, wherein the CH3 region variant comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs. 5 to 8.

[0154] In some embodiments, the polypeptide comprises one or more polypeptides containing an Fc region variant of canine IgG, wherein the Fc region variant comprises an amino acid sequence that is at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to the amino acid sequence described in any one of SEQ ID NOs: 9 to 12.

[0155] Other substitutions that can be combined with substitutions that improve half-life The development of therapeutic polypeptides / proteins (e.g., monoclonal antibodies) is a complex process that requires the coordination of a series of intricate operations to produce the desired polypeptide / protein. These include optimizing specificity, affinity, functional activity, expression levels in artificial cell lines, long-term stability, removal or enhancement of effector functions, and the development of commercially viable manufacturing and purification methods. This disclosure encompasses substitutions at one or more additional amino acid positions in Fc region variants that facilitate one or more of the above objectives.

[0156] In some embodiments, the Fc region variant includes amino acid substitutions at one or more additional amino acid positions, resulting in an increase or decrease in effector function and / or improvement in product heterogeneity.

[0157] In some embodiments, substitutions are introduced to reduce the effector function of the canine Fc region. Such substitutions can be at one or more of the following positions (numbered according to EU numbering) of canine IgG (e.g., 1, 2, 3, 4, 5, 6, or 7): 238, 265, 297, 298, 299, 327, and 329. The substitution(s) may be for any of the other 19 amino acids. In some cases, the substitutions are conserved. In a certain non-limiting example, the substituted amino acid at position 238 is Ala, the substituted amino acid at position 265 is Ala, the substituted amino acid at position 297 is Ala or Gln, the substituted amino acid at position 298 is Pro, the substituted amino acid at position 299 is Ala, the substituted amino acid at position 327 is Gly, and the substituted amino acid at position 329 is Ala. In some cases, the variant Fc region originates from a canine IgGB antibody or a canine IgGC antibody.

[0158] In some embodiments, substitutions are introduced into the Fc region of wild-type canine IgG to enhance binding to protein A, thereby facilitating purification by protein A chromatography. Such substitutions can be at one or both (e.g., 1, 2, 3, 4, 5, 6, or 7) of the canine IgG positions (numbering according to EU numbering): 252 and 254. The substitution(s) may also be for any of the other 19 amino acids. In some cases, the substitutions are conserved. In a certain non-limiting example, the substituted amino acid at position 252 is Met, and the substituted amino acid at position 254 is Ser.

[0159] In some embodiments, substitutions are made to alter the binding affinity to FcRn compared to the parent polypeptide or wild-type polypeptide (e.g., to increase or decrease the binding affinity to FcRn). In some variations, the modifications may be one, two, three, or four modifications selected from the group consisting of 308F, 428L, 434M, and 434S, with numbering following EU numbering. In some embodiments, the Fc variant includes one or more modifications selected from the group consisting of 252Y / 428L, 428L / 434H, 428L / 434F, 428L / 434Y, 428L / 434A, 428L / 434M, and 428L / 434S, with numbering following EU numbering. In some embodiments, the Fc variant includes one or more modifications selected from the group consisting of 428L / 434S and 308F / 428L / 434S, where the numbering follows EU numbering. In some embodiments, the Fc variant includes one or more modifications selected from the group consisting of 259I / 434S, 308F / 434S, 308F / 428L / 434S, 259I / 308F / 434S, 307Q / 308F / 434S, 250I / 308F / 434S, and 308F / 319L / 434S, where the numbering follows EU numbering. A detailed description of these modifications is provided, for example, in US8883973B2, which is incorporated herein by reference in its entirety.

[0160] In some embodiments, the polypeptide comprises the hinge region of the canine antibody. In some embodiments, modifications can be made to the hinge region of the canine antibody to increase its half-life. In some embodiments, the modification is 228P, following EU numbering.

[0161] In some embodiments, binding with FcRn is pH-dependent. H310 and H435 (EU numbering) may be essential for pH-dependent binding. Therefore, in some embodiments, the amino acid at position 310 (EU numbering) is histidine. In some embodiments, the amino acid at position 435 (EU numbering) is histidine. In some embodiments, the amino acids at both positions are histidine.

[0162] In some embodiments, the Fc region has LALA mutations (mutations of L234A and L235A in EU numbering) or LALA-PG mutations (mutations of L234A, L235A, and P329G in EU numbering). In some embodiments, the LALA mutation is P234A, M234A, or S234A. In some embodiments, the amino acid residue at position 234 (EU numbering) is Ala. In some embodiments, the amino acid residue at position 234 (EU numbering) is Ala. In some embodiments, the amino acid residues at positions 234 and 235 (EU numbering) are Ala.

[0163] polypeptide containing canine IgG Fc variant This disclosure encompasses any polypeptides that can benefit from increasing their half-life in dogs. To increase the half-life, these polypeptides are designed to include Fc region variants disclosed above (e.g., CH2 region, CH3 region, CH2+CH3 region).

[0164] Exemplary polypeptides include, but are not limited to, whole antibodies, scFv, nanobodies, ligand-binding moieties of receptors, cytokines, growth factors, enzymes, and peptides. For example, the CH3 domain variant disclosed above may be bound to scFv nanobodies, ligand-binding moieties of receptors (e.g., the ligand-binding moieties of canine IL-13Rα1 or IL-13Rα2), cytokines, growth factors, enzymes, or peptides. As used herein, the terms “nanobody,” “VHH,” “VHH antibody fragment,” and “single-domain antibody” are used interchangeably herein to refer to the variable domain of a single heavy chain of antibodies of the type found in camelids, which are typically naturally occurring and lack a light chain. Preferred nanobodies are well known to those skilled in the art, and examples include nanobodies of camels, dromedaries, llamas, and alpacas. Alternatively, the Fc region variant disclosed above may be bound to these polypeptides. In another embodiment, a canine or canine antibody is modified to include the Fc region variant disclosed herein.

[0165] In some embodiments, the polypeptides of this disclosure include an antibody hinge region. The hinge region may be positioned between the ligand-binding domain of the antigen or polypeptide and an Fc region variant. In some cases, the hinge region is bound to the C-terminus of a cytokine, growth factor, enzyme, or peptide, and the hinge region is bound to the N-terminus of an Fc region variant. Exemplary hinge region sequences are provided below. IgGA:FNECRCTDTPPCPVPEP(SEQ ID NO: 17); IgGB:PKRENGRVPRPPDCPKCPAPEM (Sequence ID 18); IgGC:AKECECKCNCNNCPCPGCGL(SEQ ID NO: 19); IgGD:PKESTCKCISPCPVPES(SEQ ID NO: 20); and IgGDmut:PKESTCKCIPPCPVPES (SEQ ID NO: 21).

[0166] The hinge region in the recombinant protein of this disclosure, when used, may contain 0 to 6 amino acid substitutions (i.e., 0, 1, 2, 3, 4, 5, or 6) compared to the amino acid sequence described in any one of SEQ ID NOs. 17-21. In some cases, the hinge region used in the recombinant protein of this disclosure is at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or 100% identical to the amino acid sequence described in any one of SEQ ID NOs. 17-21.

[0167] In some embodiments, the linker sequence may be used in place of the antibody hinge sequence to link a polypeptide (e.g., antibody, ligand-binding domain of a receptor, enzyme, ligand, peptide) to a canine Fc region variant disclosed herein. In certain embodiments, the linker consists of 1 to 20 amino acids linked by peptide bonds, where the amino acids are selected from 20 naturally occurring amino acids. Some of these amino acids can be glycosylated, as is well understood by those skilled in the art. In other embodiments, the 1 to 20 amino acids are selected from glycine, alanine, proline, asparagine, glutamine, and lysine. In other embodiments, the linker is largely composed of sterically unhinged amino acids such as glycine and alanine. Examples of peptide linkers include Gly, Ser;Gly Ser;Gly Gly Ser;Ser Gly Gly;Gly Gly Gly Ser (SEQ ID NO: 22);Ser Gly Gly Gly (SEQ ID NO: 23);Gly Gly Gly Gly Ser (SEQ ID NO: 24);Ser Gly Gly Gly Gly (SEQ ID NO: 25);Gly Gly Gly Gly Gly Ser (SEQ ID NO: 26);Ser Gly Gly Gly Gly Gly (SEQ ID NO: 27);Gly Gly Gly Gly Gly Gly Ser (SEQ ID NO: 28);Ser Gly Gly Gly Gly Gly Gly (SEQ ID NO: 29);(Gly Gly Gly Gly Ser) n(Sequence code 24)n (where n is an integer greater than or equal to 1 (e.g., 1, 2, 3, 4, 5)); and (Ser Gly Gly Gly Gly) n (Sequence code 25)n (where n is an integer greater than or equal to 1 (for example, 1, 2, 3, 4, 5)) is an example.

[0168] Non-peptide linkers can also be used to link one or more polypeptides of interest to the Fc region variants disclosed herein. For example, -NH(CH2) n Alkyl linkers such as C(O)-(wherein n=2~20) can be used. These alkyl linkers may be further substituted with any sterically unhindered group such as lower alkyl (e.g., C1-C6), lower acyl, halogen (e.g., Cl, Br), CN, NH2, phenyl, etc.

[0169] One or more polypeptides of this disclosure may include a binding domain. The binding domain can specifically bind to a protein, subunit, domain, motif, and / or epitope of a select target as described herein. In some embodiments, one or more polypeptides (e.g., fusion polypeptides) may include a protein, where the protein is a therapeutic protein as described herein. In some embodiments, the target (e.g., the target of the binding domain) or the therapeutic protein (e.g., fusion polypeptide) is selected from the group consisting of: 17-IA, 4-1BB, 4Dc, 6-keto-PGF1a, 8-iso-PGF2a, 8-oxo-dG, A1 adenosine receptor, A33, ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIA ALK-2, activin RIB ALK-4, activin RIIA, activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, adresin, aFGF, ALCAM, ALK, ALK-1, ALK-7, alpha-1 antitrypsin, alpha-V / beta-1 antagonist, ANG, Ang, APAF-1, APE, APJ, APP, APRIL, AR, IgE, angiotensin type 1 (AT1) receptor, angiotensin type 2 (A T2 receptor, ARC, ART, artemin, anti-Id, ASPARTIC, atrial natriuretic factor, av / b3 integrin, Axl, b2M, B7-1, B7-2, B7-H, B lymphocyte stimulating factor (BlyS), BACE, BACE-1, Bad, BAFF, BAFF-R, Bag-1, BAK, Bax, BCA-1, BCAM, Bcl, BCMA, BDNF, b-ECGF, bFGF, BID, Bik, BIM, BLC, BL-CAM, BLK, BMP, BMP-2, BMP-2a, BMP-3 osteogenin, BMP-4, BMP-2b, BMP-5, BMP-6Vgr-1, BMP-7 (OP-1), BMP-8 (BMP-8a, OP-2), BMPR, BMPR-IA (ALK-3), BMPR-IB (ALK-6), BRK-2, RPK-1, BMPR-II (BRK-3), BMPs, b-NGF, BOK, bombesin, bone-derived neurotrophic factor, BPDE, BPDE-DNA, BTC, complement factor 3 (C3), C3a, C4, C5, C5a, C10, CA125, CAD-8, calcitonin, cAMP, carcinoembryonic antigen (CEA), carcinoma-associated antigen, cathepsin A, cathepsin B, Cathepsin C / DPPI, Cathepsin D, Cathepsin E, Cathepsin H, Cathepsin L, Cathepsin O, Cathepsin S, Cathepsin V, Cathepsin X / Z / P, CBL, CC1, CCK2, CCL, CCL1, CCL11, CCL12, CCL13, CCL14, CCL15, CCL16, CCL17, CCL18, CCL19, CCL2, CCL20, CCL21, CCL22, CCL23, CCL24, CCL25, CCL26, CCL27, CCL28, CCL3, CCL4, CCL5, CCL6, CC L7, CCL8, CCL9 / 10, CCR, CCR1, CCR10, CCR10, CCR2, CCR3, CCR4, CCR5, CCR6, CCR7, CCR8, CCR9, CD1, CD2, CD3, CD3E, CD4, CD5, CD6, CD7, CD8, C D10, CD11a, CD11b, CD11c, CD13, CD14, CD15, CD16, CD18, CD19, CD20, CD21, CD22, CD23, CD25, CD27L, CD28, CD29, CD30, CD30L, CD32, CD33(p6 7 proteins), CD34, CD38, CD40, CD40L, CD44, CD45, CD46, CD47, CD49a, CD52, CD54, CD55, CD56, CD61, CD64, CD66e, CD74, CD80(B7-1), CD89, CD95, CD123, CD137, CD138, CD140a, CD146, CD147, CD148, CD152, CD164, CEACAM5, CFTR, cGMP, CINC, botulinum toxin, Clostridium perfringens toxin, CKb8-1, CLC, CMV, CMVUL, CNTF, CNTN-1, COX, C-Ret, CRG-2, CT-1, CTACK, CTGF, CTLA-4, CX3CL1, CX3CR1, CXCL, CXCL1, CXCL2, CXCL3, CXCL4, CXCL5, CXCL6, CXCL7 , CXCL8, CXCL9, CXCL10, CXCL11, CXCL12, CXCL13, CXCL14, CXCL15, CXCL16, CXCR, CXCR1, CXCR2, CXCR3, CXCR4, CXCR5, CXCR6, cytokeratin tumor-related Antigen, DAN, DCC, DcR3, DC-SIGN, Degradation Promoter, des(1-3)-IGF-I (brain IGF-1), Dhh, Digoxin, DNAM-1, Dnase, Dpp, DPPIV / CD26, Dtk, ECAD, EDA, EDA-A1, EDA-A2, EDAR, EGF, EGFR (ErbB-1), EMA, EMMPRIN, ENA, Endothelin Receptor, Enkephalinase, eNOS, Eot, Eotaxin 1, EpCAM, Ephrin B2 / EphB4, EPO, ERCC, E-Selecti N, ET-1, Factor IIa, Factor VII, Factor VIIIc, Factor IX, Fibroblast-Activating Protein (FAP), Fas, FcR1, FEN-1, Ferritin, FGF, FGF-19, FGF-2, FGF3, FGF-8, FGFR, FGFR-3, Fibrin, FL, FLIP, Flt-3, Flt-4, Follicle-Stimulating Hormone, Fractalkine, FZD1, FZD2, FZD3, FZD4, FZD5, FZD6, FZD7, FZD8, FZD9, FZD10, G250, Gas6, GCP-2, G CSF, GD2, GD3, GDF, GDF-1, GDF-3 (Vgr-2), GDF-5 (BMP-14, CDMP-1), GDF-6 (BMP-13, CDMP-2), GDF-7 (BMP-12, CDMP-3), GDF-8 (Myostatin), GDF-9, GDF-15 (MIC-1), GDNF, GDNF, GFAP, GFRa-1, GFR-Alpha 1, GFR-Alpha 2, GFR-Alpha 3, GITR, GLP1, GLP2, Glucagon, Glut4, Glycoprotein IIb / IIIa (GP IIb / IIIa), GM-CSF, gp130, gp72, GRO, GnRH, Growth hormone-releasing factor, Hapten (NP-cap or NIP-cap), HB-EGF, HCC, HCMVgB envelope glycoprotein, HCMV) gH envelope glycoprotein, HCMV UL, hematopoietic growth factor (HGF), Hep B gp120, heparanase, Her2, Her2 / neu (ErbB-2), Her3 (ErbB-3), Her4 (ErbB-4), herpes simplex virus (HSV) gB glycoprotein, HSV gD glycoprotein, HGFA, high molecular weight melanoma-associated antigen (HMW-MAA), HIV gp120, HIV IIIB gp120 V3 loop, HLA, HLA-DR, HM1.24, HMFGPEM, HRG, Hrk, cardiac myosin, cytomegalovirus (CMV), growth hormone (GH), HVEM, 1-309, IAP, ICAM, ICAM-1, ICAM-3, ICE, ICOS, IFNg, Ig, IgA receptor, IgE, IGF, IGF-binding protein, IGF-1R, IGFBP, IGF-I, IGF-II, IL, IL-1, IL-1R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL- 9, IL-10, IL-12, IL-13, IL-15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptors (e.g., IL-1R, IL-2 R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, IL-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R IL-33R), interferon (INF)-alpha, INF-beta, INF-gamma, inhibin, iNOS, insulin A chain, insulin B chain, insulin-like growth factor 1, integrin alpha 2, integrin alpha 3, integrin alpha 4, integrin alpha 4 / beta 1, integrin alpha 4 / beta 7, integrin alpha 5 (alpha V), integrin alpha 5 / beta 1, integrin alpha 5 / beta 3, integrin alpha A6, Integrin Beta 1, Integrin Beta 2, Interferon Gamma, IP-10, I-TAC, JE, Kallikrein 2, Kallikrein 5, Kallikrein 6, Kallikrein 11, Kallikrein 12, Kallikrein 14, Kallikrein 15, Kallikrein L1, Kallikrein L2, Kallikrein L3, Kallikrein L4, KC, KDR, Keratinocyte Growth Factor (KGF), Laminin 5, LAMP, LAP, LAP(TGF-1), Latent TGF-1, Latent TGF-1bp1, LBP, LDGF, LECT2, Lefty, Lewis Y antigen, Lewis Y-related antigen, LFA-1, LFA-3, Lfo, LIF, LIGHT, lipoprotein, LIX, LKN, Lptn, L-selectin, LT-a, LT-b, LTB4, LTBP-1, pulmonary surfactant, luteinizing hormone, lymphotoxin beta receptor, Mac-1, MAdCAM, MAG, MAP2, MARC, MCAM, MCAM, MCK-2, MCP, M-CSF, MDC, Mer, metalloproteinase, MGDF receptor, MGMT, MHC (HLA-DR), MIF, MIG, MIP, MIP-1-alpha, MK, MMAC1, MMP, MMP-1, MMP-10, MMP-11, MMP-12, MMP-13, MMP-14, MMP-15, MMP-2, MMP-24, MMP-3, MMP-7, MMP-8, MMP-9, MPIF, Mpo, MSK, MSP, mucin (Muc1), MUC18, Müllerian duct inhibitor, Mug, MuSK, NAIP, NAP, NAV1.7, NCAD, N-C Doherin, NCA90, NCAM, Neprilysin, Neurotrophin-3, -4, or -6, Neuroturin, Nerve Growth Factor (NGF), NGFR, NGF-Beta, nNOS, NO, NOS, Npn, NRG-3, NT, NTN, OB, OGG1, OPG, OPN, OSM, OX40L, OX40R, p150, p95, PADPr, Parathyroid Hormone, PARC, PARP, PBR, PBSF, PCAD, P-Cadherin, PCNA, PD1, PDL1, PDGF, PDG F, PDK-1, PECAM, PEM, PF4, PGE, PGF, PGI2, PGJ2, PIN, PLA2, placental alkaline phosphatase (PLAP), P1GF, PLP, PP14, proinsulin, prorelaxin, protein C, PS, PSA, PSCA, prostate-specific membrane antigen (PSMA), PTEN, PTHrp, Ptk, PTN, R51, RANK, RANKL, RANTES, RANTES, relaxin A chain, relaxin B chain, renin, respiratory syncytial virus (RSV) F, RSVFgp, Ret, Rheumatoid factor, RLIP76, RPA2, RSK, S100, SCF / KL, SDF-1, SERINE, Serum albumin, sFRP-3, Shh, SIGIRR, SK-1, SLAM, SLPI, SMAC, SMDF, SMOH, SOD, SPARC, Stat, STEAP, STEAP-II, TACE, TACI, TAG-72 (tumor-associated glycoprotein-72), TARC, TCA-3, T cell receptor (e.g., T cell receptor alpha / beta), TdT, TECK, TEM1, TEM5, TEM7, TEM8, TERT, Testicular PLAP-like alkaline phosphatase, TfR, TGF, TGF-alpha, TGF-beta, TGF-beta Pan-specific TGF-link R1(ALK-5), TGF-link R11, TGF-link RIIb, TGF-link RIII, TGF -Flip 1, TGF-factor 2, TGF-factor 3, TGF-factor 4, TGF-factor 5, activator Ck-1 Recombinant Tie, TIMP, TIQ, TMEFF2, Tmpo, TMPRSS2, TNF, TNF-protein TNF-Link 2, TNFc, TNF-RI, TNF-RII, TNFRSF10A(TRAIL R1Apo-2、DR4)、TNFRSF10B(TRAIL R2DR5、KILLER、TRICK-2A、TRICK-B)、TNFRSF10C(TRAIL R3DcR1、LIT、TRID)、TNFRSF10D(TRAIL R4 DcR2、TRUNDD)、TNFRSF11A(RANK ODF R、TRANCE R)、TNFRSF11B(OPG OCIF、TR1)、TNFRSF12(TWEAK R FN14)、TNFRSF13B(TACI)、TNFRSF13C(BAFF R)、TNFRSF14(HVEM ATAR、HveA、LIGHT R、TR2)、TNFRSF16(NGFR p75NTR)、TNFRSF17(BCMA)、TNFRSF18(GITR AITR)、TNFRSF19(TROY TAJ、TRADE)、TNFRSF19L(RELT)、TNFRSF1A(TNF). R1CD120a, p55–60, TNFRSF1B(TNF RII CD120b, p75–80), TNFRSF26(TNFRH3), TNFRSF3(LTbR TNF RIII, TNFC R), TNFRSF4(OX40 ACT35, TXGP1). R), TNFRSF5(CD40 p50), TNFRSF6(Fas Apo-1, APT1, CD95), TNFRSF6B(DcR3M68, TR6), TNFRSF7(CD27), TNFRSF8(CD30), TNFRSF9(4-1BB). CD137, ILA, TNFRSF21 (DR6), TNFRSF22 (DCTRAIL R2 TNFRH2), TNFRST23 (DCTRAIL R1TNFRH1), TNFRSF25 (DR3Apo-3, LARD, TR-3, TRAMP, WSL-1), TNFSF10 (TRAIL).Apo-2 ligand (TL2), TNFSF11 (TRANCE / RANK ligand ODF, OPG ligand), TNFSF12 (TWEAK Apo-3 ligand, DR3 ligand), TNFSF13 (APRILTALL2), TNFSF13B (BAFF BLYS, TALL1, THANK, TNFSF20), TNFSF14 (LIGHT HVEM ligand, LTg), TNFSF15 (TL1A / VEGI), TNFSF18 (GITR ligand AITR ligand, TL6), TNFSF1A (TNF-α Conectin, DIF, TNFSF2), TNFSF1B (TNF-β LTa, TNFSF1), TNFSF3 (LTb TNFC, p33), TNFSF4 (OX40 ligand gp34, TXGP1), TNFSF5 (CD40 ligand CD154, gp39, HIGM1, IMD3, TRAP), TNFSF6 (Fas ligand Apo-1 ligand, APT1 ligand), TNFSF7 (CD27 ligand CD70), TNFSF8 (CD30 ligand CD153), TNFSF9 (4-1BB ligand CD137 ligand), TP-1, t-PA, Tpo, TRAIL, TRAIL R, TRAIL-R1, TRAIL-R2, TRANCE, transfering receptor, TRF, Trk (e.g., TrkA), TROP-2, TSG, TSLP, tumor-associated antigen CA125, tumor-associated antigen expressing Lewis Y-associated carbohydrate, TWEAK, TXB2, Ung, UPAR, uPAR-1, urokinase, VCAM, VCAM-1, VECAD, VE-cadherin, VE-cadherin-2, VEFGR-1 (fit-1), VEGF, VEGFR, VEGFR-3 (flt-4), VEGI, VI M, viral antigens, VLA, VLA-1, VLA-4, VNR integrin, von Willebrand factor, WIF-1, WNT1, WNT2, WNT2B / 13, WNT3, WNT3A, WNT4, WNT5A, WNT5B, WNT6, WNT7A, WNT7B, WNT8A, WNT8B, WNT9A, WNT9A, WNT9B, WNT10A, WNT10B, WNT11, WNT16, XCL1, XCL2, XCR1, XCR1, XEDAR, XIAP, XPD, and receptors for hormones and growth factors.

[0170] In some embodiments, the binding domain specifically binds to one or more therapeutic targets or antigens in dogs, for example, but not limited to ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIAALK-2, activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, Angiotensin type 1 (AT1) receptor, Angiotensin type 2 (AT2) receptor, Atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA-4, EGFR (ErbB-1), EPO, Follicle-stimulating hormone, GDF-8 (Myostatin), GLP1, GLP2, GnRH, Growth hormone-releasing factor, IgE, IL, IL-1, IL-1 R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL -15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptors (e.g., IL -1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, I L-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP(TGF-1), latent TGF-1, latent TGF-1Examples include bp1, LFA-1, nerve growth factor (NGF), NGFR, NGF-beta, OX40L, OX40R, PD1, PDL1, TGF, TGF-alpha, TGF-beta, TGF-beta Pan-specific, TGF-beta R1 (ALK-5), TGF-beta R11, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, TNF, TNF-alpha, TNF-alpha-beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFRp75NTR), TNFRSF9 (4-1BBCD137, ILA), VEFGR-1 (fit-1), VEGF, VEGFR, and VEGFR-3 (flt-4).

[0171] In some embodiments, one or more polypeptides may include proteins, where the protein is a therapeutic protein, such as EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, a GLP-1 receptor agonist, or a thrombopoietin-binding peptide. In some embodiments, the therapeutic protein may be ACE, ACE-2, activin, activin A, activin AB, activin B, activin C, activin RIA, activin RIAALK-2, or activin RIB ALK-4, Activin RIIA, Activin RIIB, ADAM, ADAM10, ADAM12, ADAM15, ADAM17 / TACE, ADAMS, ADAM9, ADAMTS, ADAMTS4, ADAMTS5, ANG, Ang, Angiotensin type 1 (AT1) receptor, Angiotensin type 2 (AT2) receptor, Atrial natriuretic factor, av / b3 integrin, b-ECGF, CD19, CD20, CD30, CD34, CD40, CD40L, CD47, COX, CTLA-4, EGFR (ErbB-1), EPO, Follicle-stimulating hormone, GDF-8 (Myostatin), GLP1, GLP2, GnRH, Growth hormone-releasing factor, IgE, IL, IL-1, IL-1 R, IL-2, IL-2R, IL-4, IL-4R, IL-5, IL-5R, IL-6, IL-6R, IL-8, IL-9, IL-10, IL-12, IL-13, IL -15, IL-17, IL-18, IL-18R, IL-21, IL-22, IL-23, IL-25, IL-31, IL-33, interleukin receptors (e.g., IL -1R, IL-2R, IL-4R, IL-5R, IL-6R, IL-8R, IL-9R, IL-10R, IL-12R, IL-13R, IL-15R, IL-17R, I L-18R, IL-21R, IL-22R, IL-23R, IL-25R, IL-31R, IL-33R), LAP(TGF-1), latent TGF-1, latent TGF-1These include bp1, LFA-1, nerve growth factor (NGF), NGFR, NGF-beta, OX40L, OX40R, PD1, PDL1, TGF, TGF-alpha, TGF-beta, TGF-beta Pan-specific, TGF-beta R1 (ALK-5), TGF-beta R11, TGF-beta RIIb, TGF-beta RIII, TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, TGF-beta 5, TNF, TNF-alpha, TNF-alpha-beta, TNF-beta 2, TNFc, TNF-RI, TNF-RII, TNFRSF16 (NGFRp75NTR), TNFRSF9 (4-1BBCD137, ILA), VEFGR-1 (fit-1), VEGF, VEGFR, or VEGFR-3 (flt-4).

[0172] In some embodiments, the therapeutic protein is any protein described herein. In some embodiments, one or more polypeptides further comprise a canine IgG CH2 domain, IgG CH3 domain, or IgG Fc region described herein. The modified canine IgG CH2 domain, IgG CH3 domain, or IgG Fc region can increase the half-life of the therapeutic protein in vivo.

[0173] Pharmaceutical composition To prepare pharmaceutical or sterile compositions of one or more polypeptides described herein, one or more polypeptides may be mixed with pharmaceutically acceptable carriers or excipients. (See, for example, Remington's Pharmaceutical Sciences and US Pharmacopeia: National Formulary, Mack Publishing Company, Easton, Pa. (1984)).

[0174] Therapeutic and diagnostic formulations can be prepared, for example, in the form of lyophilized powders, slurries, aqueous solutions, or suspensions by mixing with acceptable carriers, excipients, or stabilizers (e.g., Hardman, et al. (2001) Goodman and Gilman's The Pharmacological Basis of Therapeutics, McGraw-Hill, New York, NY; Gennaro (2000) Remington: The Science and Practice of Pharmacy, Lippincott, Williams, and Wilkins, New York, NY; Avis, et al. (eds.) (1993) Pharmaceutical Dosage Forms: Parenteral Medications, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: Tablets, Marcel Dekker, NY; Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms Forms: Disperse Systems, Marcel Dekker, NY; Weiner and Kotkoskie (2000) Excipient Toxicity and Safety, Marcel Dekker, Inc., New York, NY (see reference). In one embodiment, one or more polypeptides of the present invention are diluted to an appropriate concentration with a sodium acetate solution at pH 5-6, and NaCl or sucrose is added for tonicity. Additional agents such as polysorbate 20 or polysorbate 80 may be added to enhance stability.

[0175] The toxicity and therapeutic efficacy of polypeptide compositions administered alone or in combination with other drugs are determined by standard pharmaceutical procedures in cell cultures or experimental animals, e.g., LD 50 (A lethal dose for 50% of the population) and ED 50This can be determined by a procedure for determining the therapeutically effective dose (LD50%) in the population. The dose-to-toxicity ratio is the therapeutic index (LD50). 50 / ED 50 In certain embodiments, one or more polypeptides exhibiting a high therapeutic index are desirable. Data obtained from these cell culture assays and animal studies can be used to derive a range of dosages for use in dogs. Doses of such compounds are preferably ED with little or no toxicity. 50 This falls within the circulating concentration range. The dosage may vary within this range depending on the dosage form and route of administration used.

[0176] The mode of administration can vary. Preferred routes of administration include oral, rectal, transmucosal, intestinal, parenteral; intramuscular, subcutaneous, intradermal, intramedullary, intrathecal, direct intraventricular, intravenous, intraperitoneal, intranasal, intraocular, inhalation, inhalation, topical, cutaneous, transdermal, or intraarterial. In some embodiments, one or more polypeptides may be administered by an invasive route such as injection. In further embodiments, one or more polypeptides may be administered intravenously, subcutaneously, intramuscularly, intraarterially, intratumorally, or by inhalation, aerosol delivery.

[0177] The pharmaceutical compositions disclosed herein may be administered by infusion. Examples of well-known forms of implants and modules for administering pharmaceutical compositions include U.S. Patent No. 4,487,603 disclosing an implantable microinfusion pump for dispensing a drug at a controlled rate; U.S. Patent No. 4,447,233 disclosing a drug infusion pump for delivering a drug at a precise infusion rate; U.S. Patent No. 4,447,224 disclosing an implantable variable flow rate infusion device for continuous drug delivery; and U.S. Patent No. 4,439,196 disclosing a permeable drug delivery system having a multi-chamber compartment. Many other such implants, delivery systems, and modules are well known to those skilled in the art.

[0178] Alternatively, one or more polypeptides may be administered topically rather than systemically, for example, by direct injection of antibodies, often in depot or sustained-release formulations, into joints or pathogen-induced lesions of arthritis characterized by immunopathology. Furthermore, one or more polypeptides may be administered via a targeted drug delivery system, for example, by liposomes coated with tissue-specific antibodies, targeting joints or pathogen-induced lesions of arthritis characterized by immunopathology. The liposomes are targeted to the affected tissue and selectively taken up by the affected tissue.

[0179] The administration regimen depends on several factors, but is not limited to, the age, weight, and physical condition of the dog being treated, the serum or tissue turnover rate of the therapeutic antibody, the level of symptoms, the immunogenicity of one or more therapeutic polypeptides, and the accessibility of target cells in the biological matrix. Preferably, the administration regimen delivers one or more therapeutic polypeptides in sufficient quantity to bring about improvement in the targeted disease condition while simultaneously minimizing undesirable side effects. Therefore, the amount of biological agent delivered depends in part on one or more specific therapeutic polypeptides and the severity of the condition being treated. Guidance on selecting the appropriate dose of therapeutic antibodies is available (see, for example, Wawrzynczak Antibody Therapy, Bios Scientific Pub. Ltd, Oxfordshire, UK (1996); Milgrom et al. New Engl. J. Med. 341:1966-1973 (1999); Slamon et al. New Engl. J. Med. 344:783-792 (2001); Beniaminovitz et al. New Engl. J. Med. 342:613-619 (2000); Ghosh et al. New Engl. J. Med. 348:24-32 (2003); Lipsky et al. New Engl. J. Med. 343:1594-1602 (2000)).

[0180] Determining the appropriate dose of one or more polypeptides is done by those skilled in the art, for example, using parameters or factors known or estimated to influence the treatment in the art. Generally, administration is started at a dose somewhat lower than the optimal dose and then increased in small increments until the desired or optimal effect is achieved against any negative side effects. Important diagnostic indicators include, for example, symptoms of inflammation or the level of inflammatory cytokines produced.

[0181] Nucleic acids, vectors, host cells, and methods for producing them. This disclosure also includes one or more nucleic acids encoding one or more polypeptides described herein, one or more vectors comprising one or more nucleic acids, and host cells comprising one or more nucleic acids or one or more vectors.

[0182] One or more polypeptides described herein may be produced in bacterial or eukaryotic cells. Some polypeptides, e.g., Fab', can be produced in bacterial cells, e.g., E. coli cells. Polypeptides can also be produced in eukaryotic cells such as transformed cell lines (e.g., CHO, 293E, COS, 293T, Hela). In addition, polypeptides (e.g., scFv) can be expressed in yeast cells such as Pichia (e.g., Powers et al., J Immunol Methods. 251:123-35 (2001)), Hanseula, or Saccharomyces. To produce the antibody of interest, one or more polynucleotides encoding one or more polypeptides are constructed, introduced into one or more expression vectors, and then expressed in suitable host cells. To improve expression, the nucleotide sequence of the gene can be recoded without altering (or making minimal changes to, e.g., removal of C-terminal residues of the heavy or light chain) the amino acid sequence. Regions that may be subject to recoding include those related to translation initiation, codon usage frequency, and unintended mRNA splicing candidates. The polynucleotides encoding the Fc region variants described herein will be readily apparent to those skilled in the art.

[0183] Using standard molecular biology techniques, recombinant expression vectors (or multiple vectors) can be prepared, host cells can be transfected, transformants can be selected, host cells can be cultured, and polypeptides (e.g., antibodies) can be recovered.

[0184] When one or more polypeptides are expressed in bacterial cells (e.g., E. coli), the expression vector must have features that allow for the amplification of the vector in the bacterial cells. Furthermore, when E. coli such as JM109, DH5α, HB101, or XL1-Blue is used as the host, the vector must have a promoter capable of enabling sufficient expression in E. coli, such as the lacZ promoter (Ward et al., 341:544-546 (1989)), the araB promoter (Better et al., Science, 240:1041-1043 (1988)), or the T7 promoter. Examples of such vectors include, for example, the M13 vector, the pUC series vectors, pBR322, pBluescript, pCR-Script, pGEX-5X-1 (Pharmacia), "QIAexpress system" (QIAGEN), pEGFP, and pET (when this expression vector is used, the host is preferably BL21 expressing T7 RNA polymerase). The expression vector may contain a signal sequence for antibody secretion. In the case of production into the periplasm of E. coli, the pelB signal sequence (Lei et al.) al., J. Bacteriol., 169:4379 (1987)) can be used as a signal sequence for antibody secretion. In the case of bacterial expression, the expression vector can be introduced into bacterial cells using the calcium chloride method or electroporation method.

[0185] When one or more polypeptides are expressed in animal cells such as CHO, COS, and NIH3T3 cells, the expression vector includes promoters necessary for expression in these cells, such as the SV40 promoter (Mulligan et al., Nature, 277:108 (1979)) (e.g., early Simian virus 40 promoter), the MMLV-LTR promoter, the EF1α promoter (Mizushima et al., Nucleic Acids Res., 18:5322 (1990)), or the CMV promoter (e.g., human cytomegalovirus pre-early promoter). In addition to the nucleic acid sequence encoding the Fc region variant, the recombinant expression vector may contain additional sequences such as sequences that regulate vector replication in the host cell (e.g., origin of replication) and selection marker genes. Selection marker genes facilitate the selection of host cells into which the vector has been introduced (e.g., U.S. Patents 4,399,216, 4,634,665, and 5,179,017). For example, typically, selection marker genes confer resistance to drugs such as G418, hygromycin, or methotrexate to host cells into which the vector has been introduced. Examples of vectors containing selection markers include pMAM, pDR2, pBK-RSV, pBK-CMV, pOPRSV, and pOP13.

[0186] In some embodiments, one or more polypeptides are produced in mammalian cells. Exemplary mammalian host cells for expressing one or more polypeptides include Chinese hamster ovary (CHO cells) (including dhfr-CHO cells described in Urlaub and Chasin (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, and used with a DHFR selection marker, for example, as described in Kaufman and Sharp (1982) Mol. Biol. 159:601621), human fetal kidney 293 cells (e.g., 293, 293E, 293T), COS cells, NIH3T3 cells, lymphocyte cell lines, for example, NS0 myeloma cells and SP2 cells, and cells derived from transgenic animals, for example, transgenic mammals. For example, cells are mammary epithelial cells.

[0187] In an exemplary system for antibody expression, a recombinant expression vector encoding both the antibody heavy and light chains is introduced into dhfr-CHO cells via calcium phosphate transfection. Within the recombinant expression vector, the antibody heavy and light chain genes are operably ligated to enhancer / promoter regulatory elements (e.g., those derived from SV40, CMV, adenovirus, etc., e.g., a CMV enhancer / AdMLP promoter regulatory element or an SV40 enhancer / AdMLP promoter regulatory element) to drive high levels of gene transcription. The recombinant expression vector also contains the DHFR gene, which allows for the selection of vector-transfected CHO cells using methotrexate selection / amplification. The selected transformed host cells are cultured to express the antibody heavy and light chains, and the antibody is recovered from the culture medium.

[0188] Treatment method One or more polypeptides disclosed herein can be used to treat or prevent any disease or disorder in dogs in need. The present invention is particularly useful for treating chronic conditions requiring repeated administration. The increased half-life of the protein therapeutic agent may allow for a reduction in the frequency of administration and / or the dose level.

[0189] In some embodiments, the diseases, disorders, conditions, or symptoms treated or prevented are allergic diseases, chronic pain, acute pain, inflammatory diseases, autoimmune diseases, endocrine disorders, gastrointestinal disorders, skeletal / musculoskeletal disorders, cardiovascular diseases, neurological disorders, kidney diseases, metabolic disorders, immune disorders, genetic / hereditary disorders, reproductive function disorders, infections, or cancer. In certain embodiments, the diseases or disorders treated or prevented are atopic dermatitis, allergic dermatitis, food allergies, osteoarthralgia, perioperative pain, toothache, cancer pain, arthritis, anemia, obesity, or diabetes.

[0190] Antibodies can be used not only to treat or prevent diseases, but also to regulate normal biological functions, for example, to manage reproductive capacity or behavior.

[0191] diagnosis One or more polypeptides disclosed herein can also be used for various diagnostic applications, for example, to determine whether a dog has any particular disease or disorder. In some embodiments, the one or more polypeptides can include a binding domain. The binding domain can specifically bind to proteins, subunits, domains, motifs, and / or epitopes (e.g., markers of cancer cells) as described herein. In some embodiments, the one or more polypeptides further include a labeling group. Generally, labeling groups can be classified into various types depending on the assay in which they are detected: a) isotopic labels that can be radioactive isotopes or heavy element isotopes; b) magnetic labels (e.g., magnetic particles); c) oxygen reduction active moieties; d) optical dyes; enzyme groups (e.g., horseradish peroxidase, β-galactosidase, luciferase, alkaline phosphatase); e) biotinylated groups; and f) predetermined polypeptide epitopes (e.g., leucine zipper pair sequences, binding sites for secondary antibodies, metal binding domains, epitope tags, etc.) recognized by secondary reporters. In some embodiments, the labeling group is attached to the antibody via spacer arms of various lengths to reduce potential steric hindrance. Various methods for labeling proteins are known in the art and can be used in practicing the present invention.

[0192] In some embodiments, the labeling group is a probe, a dye (e.g., a fluorescent dye), or a radioactive isotope (e.g., 3 H, 14 C, 22 Na, 36 Cl, 35 S, 33 P, or 125 I).

[0193] Certain labels also include optical dyes, including but not limited to chromophores, phosphors, and fluorophores, the latter of which are often specific. Fluorophores can be either "small molecule" phosphors or proteinaceous phosphors.

[0194] A fluorescent label can be any molecule that can be detected through its intrinsic fluorescent properties. Suitable fluorescent labels include fluorescein, rhodamine, tetramethylrhodamine, eosin, erythrosine, coumarin, methyl-coumarin, pyrene, malacite green, stilbene, Lucifer Yellow, Cascade Blue J, Texas Red, IAEDANS, EDANS, BODIPY FL, LC Red 640, Cy 5, Cy 5.5, LC Red 705, Oregon green, Alexa-Fluor dyes (Alexa Fluor 350, Alexa Fluor 430, Alexa Fluor 488, Alexa Fluor 546, Alexa Fluor 568, Alexa Fluor 594, Alexa Fluor 633, Alexa Fluor 660, Alexa Fluor 680), Cascade Blue, Cascade Yellow, and R-phycoerythrin (PE) (Molecular Probes, Eugene, Oreg.), FITC, rhodamine, and Texas Red This includes, but is not limited to, Cy5, Cy5.5, and Cy7 (Amersham Life Science, Pittsburgh, Pa.), including fluorophores. Suitable optical dyes, including fluorophores, are described in the Molecular Probes Handbook by Richard P. Haugland, which is incorporated herein by reference in its entirety.

[0195] Suitable protein-based fluorescent labels also include green fluorescent protein (including GFP from Renilla, Ptilosarcus, or Aequorea species) (Chalfie et al., 1994, Science 263:802-805), EGFP (Clontech Laboratories, Inc., Genbank accession number U55762), blue fluorescent protein (BFP, Quantum Biotechnologies, Inc., 1801 de Maisonneuve Blvd. West, 8th Floor, Montreal, Quebec, Canada H3H1J9; Stauber, 1998, Biotechniques 24:462-471; Heim et al., 1996, Curr. Biol. 6:178-182), improved yellow fluorescent protein (EYFP, Clontech Laboratories, Inc.), and luciferase (Ichiki et al. al., 1993, J.Immunol. 150:5408-5417), β-galactosidase (Nolan et al.) This includes, but is not limited to, U.S. Patent Nos. al., 1988, Proc. Natl. Acad. Sci. USA 85:2603-2607, and Renilla (WO92 / 15673, WO95 / 07463, WO98 / 14605, WO98 / 26277, WO99 / 49019, U.S. Patent Nos. 5,292,658, 5,418,155, 5,683,888, 5,741,668, 5,777,079, 5,804,387, 5,874,304, 5,876,995, and 5,925,558). All references cited above in this paragraph are expressly incorporated herein by reference in their entirety.

[0196] Assay Fc γ RI and FcγRIII binding: Binding to FcγRI and FcγRIII is an indicator of the ability of an antibody to mediate ADCC. To evaluate this property of an antibody, assays measuring the binding of the antibody to FcγRI and FcγRIII can be performed using methods known in the art. C1q binding:

[0197] Binding to C1q, the first component of complement, is an indicator of an antibody's ability to mediate complement-dependent cell-mediated cytotoxicity (CDC). To evaluate this property of an antibody, assays measuring the binding of an antibody to C1q can be performed using methods known in the art.

[0198] Half-life: Methods for measuring the half-life of antibodies are well known in the art. See, for example, Booth et al., MAbs, 10(7):1098-1110 (2018). Exemplary animal models include non-human primate models and transgenic mouse models. Transgenic mouse models (e.g., Tg32 or Tg276 transgenic mice) lack the mouse FcRn alpha chain and can express the human FcRn alpha transgene (e.g., under the control of a constitutive promoter). The human FcRn alpha chain can pair with the mouse β2-microglobulin protein in vivo to form a functional chimeric FcRn heterodimer. As an example, the half-life of a canine antibody can be measured by injecting the antibody into a canine model and measuring the antibody level in the serum over a certain period of time. [Examples]

[0199] Example 1: Alanine scanning mutation introduction into the CH2 and CH3 domains of canine IgGB. Alanine scanning mutagenesis (Morrison and Weiss, Curr. Opin. Chem. Biol. 5:302-307 (2001)) was performed on residues 250, 251, 252, 254, 256, 285, 286, 307, 309, 311, and 315 of the CH2 domain, and residues 378, 380, 428, 430, 433, 434, 435, and 436 of the CH3 domain. For this experiment, wild-type (wt) sequences of the CH2 and CH3 domains of canine IgGB were synthesized and used as templates for mutagenesis. Each specific position, except for position 254, was individually changed to alanine by PCR mutagenesis using primers encoding the change. Position 254, which is alanine in the wild-type sequence, was changed to serine. PCR products were subcloned into GenScript FASEBA plasmids, transformed into E. coli, and sequenced for the presence of variants. Upstream of the CH2 domain is a SASA (single-domain antibody against serum albumin) tag with pM affinity for albumin (see, e.g., US2013 / 0129727A1). The PelB (pectin lyase B) signal peptide is located at the N-terminus and promotes the secretion of Fc into the culture medium. CH2-CH3 protein expression was regulated by the Lac promoter. Supernatants from conditioned medium were analyzed using surface plasmon resonance (SPR) for binding to canine FcRn (UniProtKB-E2R0L6[FcRn] and UniProtKB-E2RN10[canine beta-2-microglobulin]) at pH 5.5.

[0200] In SPR analysis using Biacore 8K, bovine serum albumin (BSA) was immobilized on a CM5 sensor tip. The sensor tip surfaces of flow cells 1 and 2 were activated over 420 seconds (10 μL / min) with a freshly mixed 50 mmol / L N-hydroxysuccinimide and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Subsequently, BSA diluted with 10 mM sodium acetate (pH 4.5) was injected into flow cell 2 to achieve conjugation, and flow cell 1 was used as a blank. After the amine coupling reaction, the active coupling site remaining on the tip surface was blocked by injecting 1 mM ethanolamine hydrochloride for 420 seconds. The running buffer for the coupling experiment was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 5.5), and the experiment was performed at 25°C. The supernatant of the alanine variant was injected onto the chip surface and captured by the immobilized BSA via a SASA tag over 60 seconds. 400 nM canine FcRn was injected for 120 seconds, and dissociation was performed with running buffer for 120 seconds. The flow rate during the BSA immobilization phase was 10 μl / min, and the flow rate during the association and dissociation phases was 30 μl / min. All data were processed using Biacore 8K evaluation software version 1.1. The data in tabular form is shown in Table 3. The last column contains the mean KD of the wild type divided by the variant KD. Sensorgrams are shown in Figures 7A-7U. [Table 3-1] [Table 3-2]

[0201] Example 2: Generation of an NNK saturated mutation library at a selected location and analysis of individual variants. NNK saturated mutagenesis is an effective strategy for generating all 20 possible amino acids at desired positions (Hogrefe et al., Biotechniques. 33:1158-1165 (2002)). Individual NNK libraries were generated at positions 250, 252, 254, 309, 311, 378, 380, and 434 (EU numbering). For this method, NNK (N=A / C / G / T, K=G / T) primers at specific positions were used with the QuikChange Site-Directed Mutagenesis Kit (Agilent). The supernatants of 90 individual transformants obtained from each library were assayed for binding to canine FcRn at pH 5.5 using the Biacore method described in Example 1. The only difference was that the concentration of canine FcRn used in the assay was 200 nM instead of 400 nM. All sensorgrams of the NNK library variants are shown in Figures 8–15.

[0202] For the NNK library at position 250, none of the variants showed increased binding to canine FcRn at pH 5.5. Data for variants T250E and T250Q, as well as wild-type Fc, are shown in Table 4. In competitive binding assays, human IgG2 variants T250E and T250Q were shown to bind more strongly to human FcRn at pH 6.0 compared to wild-type human IgG2Fc (Hinton et al., J. Biol. Chem. 279:6213-6216 (2004)). [Table 4]

[0203] For the NNK library at position 252, only the L252Y and L252M variants showed apparent high affinity for canine FcRn at pH 5.5 (see Table 5 below). Since the L252F variant was not present in any of the 90 transformants, binding data for this variant could not be obtained. [Table 5]

[0204] For the NNK library at position 254, none of the variants tested showed apparent high affinity for canine FcRn at pH 5.5. Data for the A254T variant are shown in Table 6. For the corresponding human IgG1 variant, the YTE variant (M252Y / S254T / T256E) is used, which shows increased affinity for human FcRn at pH 6.0 (Dall'Acqua et al., J.Immunol.169:5171-5180 (2002)) and has been shown to increase the half-life of human IgG in preclinical models and in humans (Borrok et al., J.Biol.Chem.290:4282-4290 (2015); Robbie et al., Antimicrob.Agents Ch.57:6147-6153 (2013)). Since the A254H variant was not present in any of the 90 transformants, data for this variant could not be obtained. [Table 6]

[0205] For the NNK library at positions 309 and 311, none of the tested variants showed apparent high affinity for canine FcRn at pH 5.5. Data for variants G309P and Q311V are shown in Tables 7 and 8. The corresponding human variants of human IgG1 (L309P and Q311V) have been shown to have higher affinity for human FcRn at pH 6.0 in some combinations with other variants (Dall'Acqua et al., J.Immunol. 169:5171-5180 (2002); Booth et al., MAbs, 10(7):1098-1110 (2018)). Variants G309D, G309K, and Q311D were not identified in the NNK library and therefore were not tested for FcRn binding. [Table 7] [Table 8]

[0206] For the NNK libraries at positions 378 and 380, none of the tested variants showed apparent high affinity to canine FcRn at pH 5.5. Data for variant D378V are shown in Table 9. The corresponding human IgG1 variant used in combination with other IgG variants showed higher affinity to human FcRn at pH 6.0 compared to wild-type Fc, and extended the half-life of human IgG in transgenic human FcRn mice (Monnet et al., MABS.6:422-436(2014); Booth et al., 2018). Data for variant E380A are also shown in Table 10. The corresponding human IgG variant has been shown to have higher binding affinity to human FcRn at pH 6.0 (Shields et al., J.Biol.Chem.276:6591-6604(2001)). Variants D378E, D378I, D378K, and E380F were not present in the NNK library and were not screened for binding to canine FcRn. [Table 9] [Table 10]

[0207] For the NNK library at position 434, as shown in Table 11, variants N434Y, N434W, and N434R exhibited higher affinity for canine FcRn at pH 5.5. Variants N434S and N434A, unlike their corresponding human IgG1 variants, did not exhibit higher affinity for canine FcRn at lower pH levels (Petkova et al., Int.Immunol.18:1759-1769(2006); Yeung et al., J.Immunol.182:7663-7671(2009); Zalevsky et al., Nat.Biotechnol.28:157-159(2010); Deng et al., Drug Metab.Dispos.38:600-605(2010)). The NNK library screened at position 434 did not contain the N434F variant; therefore, the binding of this variant to canine FcRn was not tested. [Table 11]

[0208] Example 3: Binding kinetics of L252Y, N434Y, N434W, N434R, N434H and YTE (L252Y / A254T / T256E) variants and wild-type Fc For several canine IgGB variants that showed higher affinity for canine FcRn at pH 5.5, the binding kinetics to canine FcRn were further evaluated. In this study, the binding of variants (L252Y, N434Y, N434W, N434R, N434H), YTE variants (L252Y / A254T / T256E), and wild-type canine Fc to canine FcRn was evaluated at pH 5.5 and pH 7.4. The Biacore method at pH 5.5 was the same as described in Example 1, except that four concentrations of FcRn (100 nM, 200 nM, 400 nM, 800 nM) were tested, which allowed for more accurate binding kinetics. Under Biacore conditions at pH 7.4, a running buffer of 10 mM HEPES, 500 mM NaCl, 3 mM EDTA, and 0.05% Tween 20 (pH 7.4) was used, and the test concentration of canine FcRn was 200 nM. None of the variants (including YTE) and the wild type bound to canine FcRn at pH 7.4. Binding kinetics at pH 5.5 are shown in Table 12, and sensorgrams are shown in Figures 16A-16E. The tested variants showed increased affinity for canine FcRn at pH 5.5 compared to wild-type Fc. [Table 12]

[0209] Example 4: Generation of an NNK saturated mutation library at a selected location and analysis of individual variants. Wild-type (wt) sequences of the CH2 and CH3 domains of canine IgGB (SEQ ID NO: 10) were synthesized and used as templates for NNK mutagenesis. NNK saturated mutagenesis is an effective strategy for generating all 20 possible amino acids at the desired position (Hogrefe et al., Biotechniques. 33:1158-1165 (2002)). Individual NNK libraries were generated at positions 286, 312, 426, and 436 (EU numbering). NNK (N=A / C / G / T, K=G / T) primers at specific positions were used with the QuikChange Site-Directed Mutagenesis Kit (Agilent). PCR products were subcloned into GenScript FASEBA plasmids, transformed into E. coli, and sequenced for the presence of variants. Upstream of the CH2 domain is a SASA (single-domain antibody against serum albumin) tag with pM affinity for albumin (Zhang, J.; Wu, S.; Liu, J. Methods and systems for increasing protein stability. 2013 U.S. Patent Application). The SASA antibody enables the capture of Fc to the sensor chip surface as described below. The PelB (pectin lyase B) signal peptide is located at the N-terminus and promotes the secretion of Fc into the culture medium. CH2-CH3 protein expression was controlled by the Lac promoter. Supernatants from conditioned medium were analyzed for binding to canine FcRn (UniProtKB-E2R0L6[FcRn] and UniProtKB-E2RN10[canine beta-2-microglobulin]) using surface plasmon resonance (SPR) at pH 5.5 for variants at positions 426 and 312, and at pH 6.0 for variants at positions 286 and 436.

[0210] The supernatants of 90 individual transformants obtained from each library were assayed for binding to canine FcRn using the Biacore method as described below, at pH 5.5 for variants at positions 426 and 312, and at pH 6.0 for variants at positions 286 and 436.

[0211] In SPR analysis using Biacore 8K, bovine serum albumin (BSA) was immobilized on a CM5 sensor tip. The sensor tip surfaces of flow cells 1 and 2 were activated over 420 seconds (10 μL / min) with a freshly mixed 50 mmol / L N-hydroxysuccinimide and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride. Subsequently, BSA diluted with 10 mM sodium acetate (pH 4.5) was injected into flow cell 2 to achieve conjugation, and flow cell 1 was used as a blank. After the amine coupling reaction, the active coupling site remaining on the tip surface was blocked by injecting 1 mM ethanolamine hydrochloride for 420 seconds. The running buffer for the coupling experiment was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 5.5), and the experiment was performed at 25°C. The supernatant of the variant was injected onto the chip surface and captured by the immobilized BSA via a SASA tag over 60 seconds. 200 nM canine FcRn was injected for 120 seconds, and dissociation was performed with running buffer for 120 seconds. The flow rate during the BSA immobilization phase was 10 μl / min, and the flow rate during the association and dissociation phases was 30 μl / min. All data were processed using Biacore 8K evaluation software version 1.1. See Figure 17 for the Biacore sensorgram.

[0212] The tested variants showed increased binding affinity to canine FcRn at pH 5.5 (for variants containing amino acid substitutions at position 312 or 426) and pH 6.0 (for variants containing amino acid substitutions at position 286 or 436) compared to wild-type canine IgGB Fc (SEQ ID NO: 10). The results are summarized in Tables 13 and 14 below. [Table 13] [Table 14]

[0213] Example 5: Binding kinetics of A426Y, A426H, A426F, T286Y, T286F, T286L, T286W, Y436H and wild-type Fc For several canine IgGB variants that showed higher affinity for canine FcRn, the binding kinetics to canine FcRn were further evaluated. In this study, the binding of Fc to canine FcRn was evaluated for variants (A426Y, A426H, A426Y, T286Y, T286F, T286L, T286W, Y436H), YTE variants (L252Y / A254T / T256E), and wild-type canine IgGB at either pH 5.5 or pH 6.0 and at pH 7.4. The Biacore method at pH 5.5 and pH 6.0 was the same as described in Example 4 above, except that four concentrations of FcRn (100 nM, 200 nM, 400 nM, 800 nM) were tested, which allowed for more accurate binding kinetics. Under Biacore conditions at pH 7.4, a running buffer of 10 mM HEPES, 500 mM NaCl, 3 mM EDTA, and 0.05% Tween 20 (pH 7.4) was used, and the test concentration of canine FcRn was 200 nM. See Figure 18 for the Biacore sensorgram. Neither wild-type Fc nor any of the variants bound to FcRn under the conditions described at pH 7.4.

[0214] The binding affinity data is shown in Tables 15 and 16 below. [Table 15] [Table 16]

[0215] Example 6: Canine Fc variant having an amino acid substitution at position 426 of Fc in canine IgGA Two canine Fc variants possessing an amino acid modification at position 426 (according to EU numbering) and the Fc of wild-type canine IgGA (SEQ ID NO: 9) were synthesized using the variable domain described by Gearing DP et al. (2013, BMC Veterinary Research, 9:226). Canine IgGA DNA was synthesized, subcloned into the pcDNA3.4 vector (ThermoFisher), and transfected into ExpiCHO-S cells using the ExpiCHO transfection method (ThermoFisher). Fourteen days after cell transfection, the conditioned medium was purified using GenScript protein G resin.

[0216] For the binding experiment at pH 6.0, the antibody was directly conjugated to the CM5 sensor chip, and then canine FcRn was flowed through HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 6.0). The sensor chip surfaces of flow cells 1 and 2 were activated for 100 seconds (10 μL / min) with a freshly mixed 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). The antibody was diluted with 10 mmol / L NaAC (pH 4.5) and injected into flow cell 2 to achieve a conjugation of approximately 100 response units, while flow cell 1 was left blank. After the amine coupling reaction, the active coupling site remaining on the chip surface was blocked by injecting 1 mol / L ethanolamine hydrochloride for 100 seconds. The running buffer for the binding experiment at pH 6.0 was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween20, pH 6.0), and the experiment was conducted at 25°C. Canine FcRn (UniProtKB-E2R0L6[FcRn] and UniProtKB-E2RN10[Canine Beta-2-Microglobulin]) was injected for 120 seconds, and dissociation was performed in the running buffer for 120 seconds. The flow rate was 30 μL / min. The concentrations of canine FcRn injected onto the sensor chip were 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM. All data were processed using Biacore 8K evaluation software version 1.1. The injection of flow cell 1 and buffer only for each cycle was used as a reference for the response unit subtraction. Table 17 below provides kinetic data from coupling experiments at pH 6.0. Previous studies have shown that amine coupling of IgG to the Biacore CM5 biosensor chip results in a 2-3-fold decrease in affinity for FcRn compared to the affinity determined by solution-based methods or direct coupling to the Biacore C1 chip (Abdiche et al., 2015. mAbs, 7:331).Therefore, the true affinity of these IgGs to FcRn at pH 6.0 appears to be at least twice as high. However, this method is useful for comparing the relative FcRn binding affinity of different IgG Fc variants. A Biacore sensorgram is shown in Figure 19. [Table 17]

[0217] Example 7: Screening of canine IgGB Fc variants with increased FcRn binding compared to wild-type canine IgGB Fc. Canine Fc variants containing a single amino acid substitution or a combination of amino acid substitutions were synthesized in canine IgGB (SEQ ID NO: 10) format using a variable domain described by Gearing DP et al. (2013, BMC Veterinary Research, 9:226). Canine IgGB DNA was synthesized, subcloned into a pcDNA3.4 vector (ThermoFisher), and transfected into ExpiCHO-S cells using the ExpiCHO transfection method (ThermoFisher). Fourteen days after cell transfection, the conditioned medium was purified using Monofinity A resin (GenScript). Antibody binding to canine FcRn was measured using Biacore 8K under both pH 6.0 and pH 7.4 conditions.

[0218] To achieve binding conditions at pH 6.0, the sensor tip surfaces of flow cells 1 and 2 were activated for 100 seconds (10 μL / min) with a freshly mixed 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). The antibody was diluted with 10 mmol / L NaAC (pH 4.5) and injected into flow cell 2 to achieve a conjugation of approximately 100 response units, while flow cell 1 was left blank. After the amine coupling reaction, the active coupling site remaining on the tip surface was blocked by injecting 1 mol / L ethanolamine hydrochloride for 100 seconds. The running buffer for the pH 6.0 binding experiment was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 6.0), and the experiment was conducted at 25°C. Canine FcRn (UniProtKB-E2R0L6[FcRn] and UniProtKB-E2RN10[Canine Beta-2-Microglobulin]) were injected for 120 seconds, and dissociation was performed in running buffer for 120 seconds. The flow rate was 30 μL / min. The concentrations of canine FcRn injected onto the sensor chip were 200 nM, 400 nM, 800 nM, 1600 nM, and 3200 nM for wild-type IgG and A426H-N434R IgG variants. For the remaining variants, the concentrations of canine FcRn injected were 50 nM, 100 nM, 200 nM, 400 nM, and 800 nM. All data were processed using Biacore 8K evaluation software version 1.1.

[0219] The injection of only flow cell 1 and buffer in each cycle was used as the criterion for response unit subtraction. Table 18 below shows the kinetic data of the binding experiment at pH 6.0. It has been previously shown that amine coupling of IgG to the Biacore CM5 biosensor chip reduces the affinity to FcRn by 2-3 times compared to the affinity determined by solution-based methods or direct coupling to the Biacore C1 chip (Abdiche et al., 2015. mAbs, 7:331). Therefore, the true affinity of these IgGs to FcRn at pH 6.0 is likely to be at least 2 times higher. However, this method is useful for comparing the relative FcRn binding affinities of different IgG Fc variants. Biacore sensorgrams are shown in Figures 20-23. [Table 18]

[0220] To achieve the binding conditions at pH 7.4, the sensor tip surfaces of flow cells 1 and 2 were activated for 420 seconds (10 μL / min) with a freshly mixed 50 mmol / L N-hydroxysuccinimide (NHS) and 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC). Subsequently, canine FcRn diluted with 10 mmol / L NaAC (pH 4.5) was injected into flow cell 2 to achieve a conjugation of approximately 2000 response units, while flow cell 1 was left blank. After the amine coupling reaction, the active coupling site remaining on the tip surface was blocked by injecting 1 mol / L ethanolamine hydrochloride for 420 seconds. The running buffer for the pH 7.4 binding experiment was HBS-EP (10 mM HEPES, 500 mM NaCl, 3 mM EDTA, 0.05% Tween 20, pH 7.4), and the experiment was conducted at 25°C. Different antibodies were injected at 400 nM for 120 seconds, and dissociation was performed in running buffer for 120 seconds. The flow rate was 30 μL / min. Table 19 below shows the kinetic data of the binding experiment at pH 7.4. Biacore sensorgrams are shown in Figures 24-27. [Table 19]

[0221] The affinity of canine IgG Fc and canine FcRn interaction at pH 7.4 is very weak and difficult to measure using SPR with many methods. To compare the affinity of various canine Fc variants to canine FcRn at pH 7.4, a high concentration of canine FcRn was coated onto a sensor tip, and the interaction was measured by flowing variant IgG Fc onto the tip. Due to the abidite effect in this format, the measured binding affinity is not an accurate measurement of the interaction between individual canine IgG Fc variants and canine FcRn, but it can be used to compare the relative binding of variant IgG at pH 7.4. These should not be used to make a direct comparison with the binding affinity at pH 6.0.

[0222] Example 8: In vivo screening of canine IgGB Fc variants having increased FcRn binding compared to wild-type canine IgGB Fc. A pharmacokinetic (PK) study was conducted using 16 male and female beagles. Canine IgGB Fc variants containing a single amino acid substitution or a combination of amino acid substitutions were prepared by incorporating amino acid substitutions (multiple substitutions) into canine IgGB (SEQ ID NO: 10) using the anti-NGF variable domain described by Gearing DP et al. (2013, BMC Veterinary Research, 9:226; its contents are incorporated herein by reference in their entirety). The animals were randomly divided into eight groups, with males and females in each group. The mean age of the beagles was over 6 months, and their body weight was 8-10 kg. Each animal received a single intravenous dose of 2 mg / kg of antibody. Approximately 1.5 ml of whole blood was collected at the following time points: 0 (before administration), 4 hours after injection, and on days 1, 2, 4, 6, 10, 14, 18, 22, 30, 34, 38, and 42. Serum was isolated from whole blood, and the presence of antibody variants was assayed using an ELISA specific to anti-NGF antibodies.

[0223] Non-compartmental PK analysis (NCA) of individual serum antibody measurements was performed using a well-established Excel plug-in software tool for pharmacokinetic analysis ("PKSolver: An add-in program for pharmacokinetic and pharmacodynamic data analysis in Microsoft Excel", Yong Zhang et al., Comput.Methods Programs Biomed.; 2010 Sep; 99(3): 306-14. doi: 10.1016 / j.cmpb.2010.01.007; its contents are incorporated herein by reference in their entirety). PKSolver offers many options for determining PK parameters. NCA has been identified as the optimal method due to its ease of use, model independence, and ability to enhance interanalytical consistency. In a specific analysis, PKSolver's NCA IV Bolus was used to determine the terminal phase half-life (T1 / 2). Following an initial series of dose-setting experiments, measurements significantly above LLOQ were obtained up to 42 days after intravenous administration. Therefore, the terminal phase half-life could be reliably estimated by estimating the gradient for antibody measurements over at least the last four weeks. No data points were discarded or deleted in any of the experiments. As shown in Figure 29, combinations of amino acid substitutions in the IgG Fc region significantly improved the terminal phase half-life of canine anti-NGF IgGB antibodies in vivo compared to anti-NGF IgGB antibodies possessing (i) wild-type canine IgGB Fc region or (ii) canine IgGB Fc variants containing only a single amino acid substitution.

[0224] Example 9: Binding kinetics of canine IgGB variants to canine FcRn using a C1 biosensor The binding kinetics of several canine IgGB variants (A426Y, A426Y+T286L, A426Y+D312P, A426Y+Y436H, A426Y+T286L+Y436H, A426H, A426H+T286L, A426H+T286Y, A426H+D312P, A426H+Y436H, and wild-type) to canine FcRn (UniProtKB-E2R0L6 [canine macro subunit FcRn] and UniProtKB-E2RN10 [canine beta-2-microglobulin]) at pH 5.9 were evaluated. EU numbering was used for location identification (Figure 28). In this study, canine Fc variants containing a single amino acid substitution or a combination of amino acid substitutions were synthesized in canine IgGB (GENBANK accession number AAL35302.1) format using a variable domain described by Gearing DP et al. (2013, BMC Veterinary Research, 9:226). The synthesized canine IgGB DNA was subcloned into a mammalian expression vector and transiently transfected into CHO cells. The conditioned medium was purified using protein A chromatography.

[0225] For the canine FcRn binding experiments, all assays were performed at 25°C using a Biacore 8K+ system. In the experiments above (e.g., Examples 6 and 7), the affinity of IgG variants for canine FcRn was measured by amine coupling of IgG to a Biacore CM5 biosensor chip, which Abdiche et al., 2015 (mAbs, 7:331) has shown to underestimate the affinity of Fc variants for FcRn compared to when using a Series S C1 biosensor. In this experimental set, to obtain a more accurate measurement of FcRn affinity, all antibodies were immobilized on a Series S C1 sensor chip using standard amine coupling reagents. The surface was activated by injecting a mixture of 200 mmol / L 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) and 50 mmol / L N-hydroxysuccinimide (NHS) for 420 seconds. Next, the antibody was injected at a concentration of 0.5–2 μg / ml in 10 mM sodium acetate (pH 5.0) for 120 seconds. Finally, 1 M ethanolamine was injected for 420 seconds. The running buffer was 1XPBS-P+ (Cytiva, Cat#28995084) adjusted to pH 5.9.

[0226] To evaluate the binding affinity of canine IgGB variants to canine FcRn at pH 5.9, canine FcRn in the concentration range of 1.56 to 2000 nM was selected and injected in single-cycle mode. The concentrations of canine FcRn tested for each variant are shown in Table 20 below. [Table 20]

[0227] Four concentrations of antibody were injected at 5 μl / min for 90 seconds, followed by a 180-second dissociation. Each concentration series was injected three times in this format, with at least three buffer-only cycles to ensure proper reference deduction. The surface was regenerated by injecting 1X PBS-P+ (pH 7.4) twice for 30 seconds, followed by a 60-second wait command. Three startup cycles were included to stabilize the surface before analysis.

[0228] Using Insight Evaluation Software, data were evaluated by fitting to a 1:1 kinetic interaction model or to steady-state affinity. Acceptable parameters were selected using quality indices, including U and T values. U values ​​less than 15 were considered acceptable as reaction rate constants, and T values ​​greater than 100 were considered acceptable as reaction rate constants. If these values ​​were outside this range, the steady-state affinity parameters were considered acceptable.

[0229] The kinetic data for the 10 variants are shown in Table 21 below, and the sensorgrams are shown in Figures 30A to 30K. [Table 21]

[0230] Example 10: Pharmacokinetic study of canine IgGB variant with increased FcRn binding and wild-type canine IgGB. Three pharmacokinetic (PK) studies were conducted in male and female beagles. Canine Fc variants containing a single amino acid substitution or a combination of amino acid substitutions were synthesized in canine IgGB (SEQ ID NO: 10) format using the anti-NGF variable domain described by Gearing DP et al. (2013, BMC Veterinary Research, 9:226). For each study, animals were randomly assigned so that each group contained an equal number of males and females. The IgGB variants evaluated in each study and the number of males (M) and females (F) in each group are shown below (Table 22). [Table 22]

[0231] The dogs had an average age of over 6 months and weighed 8-10 kg. Each animal received a single intravenous injection of 1 mg / kg (Study 1) or 2 mg / kg (Studies 2 and 3) of antibody. Approximately 1.5 ml of whole blood was collected at the following time points: 0 (before administration), 4 hours after injection, and on days 1, 2, 4, 6, 10, 14, 18, 22, 30, 34, 38, and 42. Serum was extracted from the blood and antibody variants were assayed by NGF antibody-specific ELISA.

[0232] A nonlinear mixed-effects model was used to describe serum concentrations in a two-compartment pharmacokinetic (PK) model with linear clearance (Figure 31). Population PK parameters were estimated using the stochastic expectation maximization (SAEM) algorithm implemented in Monolix Suite 2019R1 (Monolix version 2019R1. Antony, France: LixoftSAS, 2019). Individual parameters were modeled as random variables following a log-normal distribution. Population parameters were estimated from pooled data including all variants and trials. Trials were a categorical covariate for clearance. mAb variants were identified using categorical covariates for clearance and central and peripheral volume of distribution. Categorical trial and variant covariates were described as follows:

number

[0233] In the formula, if each covariate belongs to that category, then Ω i =1 if present, otherwise Ω i = 0. The wild-type IgGB variant was used as the reference.

[0234] Figure 29 shows the terminal phase half-lives of the wild-type, A426Y, and A426Y+Y436H variants observed in Experiment 2.

[0235] Using data from all three trials, estimated PK parameters were generated for each variant (Table 23). [Table 23]

[0236] Figure 32B shows the serum concentrations of wild-type, A426Y, A426Y+Y436H, and A426Y+Y436H+T286L observed individually from Experiment 3. Figure 32A shows the serum concentrations of wild-type, YTE, N434Y, and N434R observed individually from Experiment 1.

[0237] We simulated the predicted serum concentration profiles over 3 months for anti-NGF antibodies containing wild-type IgGB Fc or IgGB variants A426Y, A426Y+Y436H, A426Y+Y436H+T286L, N434R, N434Y, and YTE. These are shown in Figure 33. In this simulation, 10 kg dogs were used, and a single intravenous dose of 2 mg / kg was administered.

[0238] Example 11: Modeling of canine IgGB Fc variants that bind to canine FcRn To gain insight into the molecular mechanism of the canine IgGB Fc variant that binds to canine FcRn, a structural model of the canine IgGB Fc-canine FcRn complex was generated using MOE software (Molecular Operating Environment (MOE), 2020.09; Chemical Computing Group ULC, 1010 Sherbrooke St. West, Suite #910, Montreal, QC, Canada, H3A 2R7, 2020) based on the cocrystal structure of the human FcRn-YTE-Fc domain complex (PDB ID: 4N0U). Mutations were incorporated into the MOE-modeled structure, and the energy was minimized using the Amber14:EHT force field. The canine Fc-FcRn interaction distance was measured using PyMOL software (The PyMOL Molecular Graphics System, Version 1.2r3pre, Schrodinger, LLC).

[0239] Figure 34 shows canine Fc positions 286, 426, and 436, which have variants that increase affinity to canine FcRn at low pH.

[0240] The canine IgGB A426H variant is shown in Figure 35. Position 426 is too far away to directly interact with FcRn. This model predicts that A426H undergoes a steric collision with Y436, changing Y436 to a favorable conformation by binding to FcRn. Canine A426Y is shown in Figure 36. Similar to A426H, it is too far away to directly interact with FcRn, and it shifts Y436 to a favorable conformation by binding to FcRn.

[0241] The canine IgGB Y436H variant is shown in Figure 37. Changing the residue at position 436 to His is expected to slightly alter the adjacent residues. The charge difference is thought to promote stronger binding. H436, like the hydrophobic / aromatic Y436, lacks charge at neutral pH, and is therefore expected to drive adjacent residues into an unfavorable environment for binding. However, the protonated His436, being hydrophilic / positively charged, provides a more attractive interface for residues such as E135 in the large FcRn subunit.

[0242] The canine IgGB T286L variant is shown in Figure 38. This does not directly interact with canine beta-2-microglobulin in FcRn. However, the existing hydrophobic interaction can be enhanced by changing threonine to leucine. This observation is consistent with the increased affinity of the T286Y, T286F, and T286W variants for canine FcRn at low pH, as shown in Examples 4 and 5.

[0243] The combination of A426Y, Y436H, and T286L variants in canine IgGB Fc was modeled (Figure 39). Due to steric collisions between the A426Y and Y436H variants, it is predicted that 436 will move to a preferred position upon binding. This may be additive with the pH-dependent effect of His. T286L appears to be too far removed to be directly affected by the mutations in 426 and 436. The in vitro binding data of the triple variant (A426Y, Y436H, and T286L) to canine FcRn at low pH in Example 9 are consistent with the model in which the combination of the three variants additively increases FcRn affinity. (Note) (Note 1) A polypeptide comprising a canine IgG Fc region variant or its canine FcRn binding region, wherein the position is selected from the group consisting of the following: (i) The position corresponding to amino acid position 286 of wild-type canine IgG; (ii) The position corresponding to amino acid position 312 of wild-type canine IgG; (iii) the position corresponding to amino acid position 426 of wild-type canine IgG; and (iv) Position corresponding to amino acid position 436 of wild-type canine IgG It contains at least one amino acid substitution, Here, the amino acid substitution corresponding to amino acid position 286 of the wild-type canine IgG is selected from the group consisting of Tyr, Phe, Leu, and Trp, the amino acid position is based on EU numbering, and the polypeptide has increased binding affinity to canine FcRn compared to the Fc domain of the wild-type canine IgG. (Note 2) The polypeptide according to Appendix 1, wherein the at least one amino acid substitution includes an amino acid substitution at a position corresponding to amino acid position 312 of wild-type canine IgG. (Note 3) The polypeptide according to Appendix 2, wherein the polypeptide contains Pro at the amino acid position corresponding to amino acid position 312 of the wild-type canine IgG. (Note 4) The polypeptide according to any one of the appendices 1 to 3, wherein the at least one amino acid substitution includes an amino acid substitution at a position corresponding to amino acid position 426 of wild-type canine IgG. (Note 5) The polypeptide according to Appendix 4, wherein the polypeptide contains Tyr, His, or Phe at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG. (Note 6) The polypeptide according to Appendix 5, wherein the polypeptide contains Tyr at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG. (Note 7) The polypeptide according to Appendix 5, wherein the polypeptide contains His at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG. (Note 8) The polypeptide according to Appendix 5, wherein the polypeptide contains Phe at the amino acid position corresponding to amino acid position 426 of the wild-type canine IgG. (Note 9) The polypeptide according to any one of the appendices 1 to 8, wherein the at least one amino acid substitution includes an amino acid substitution at a position corresponding to amino acid position 286 of wild-type canine IgG. (Note 10) The polypeptide according to Appendix 9, wherein the polypeptide contains Tyr at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG. (Note 11) The polypeptide according to Appendix 9, wherein the polypeptide contains Phe at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG. (Note 12) The polypeptide according to Appendix 9, wherein the polypeptide contains Leu at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG. (Note 13) The polypeptide according to Appendix 9, wherein the polypeptide contains Trp at the amino acid position corresponding to amino acid position 286 of the wild-type canine IgG. (Note 14) The polypeptide according to any one of the appendices 1 to 13, wherein the at least one amino acid substitution includes an amino acid substitution at a position corresponding to amino acid position 436 of wild-type canine IgG. (Note 15) The polypeptide according to Appendix 14, wherein the polypeptide contains His at the amino acid position corresponding to amino acid position 436 of the wild-type canine IgG. (Note 16) The polypeptide according to any one of the appendices 1 to 15, wherein the polypeptide contains an amino acid sequence that is at least 80% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 12. (Note 17) The position is selected from the following group: (i) The amino acid position corresponding to amino acid position 250 of the wild-type canine IgG, (ii) The amino acid position corresponding to amino acid position 251 of the wild-type canine IgG, (iii) The amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, (iv) The amino acid position corresponding to amino acid position 254 of the wild-type canine IgG, (v) The amino acid position corresponding to amino acid position 256 of the wild-type canine IgG, (vi) The amino acid position corresponding to amino acid position 285 of the wild-type canine IgG, (vii) The amino acid position corresponding to amino acid position 286 of the wild-type canine IgG, (viii) The amino acid position corresponding to amino acid position 307 of the wild-type canine IgG, (ix) The amino acid position corresponding to amino acid position 308 of the wild-type canine IgG, (x) The amino acid position corresponding to amino acid position 309 of the wild-type canine IgG, (xi) The amino acid position corresponding to amino acid position 311 of the wild-type canine IgG, (xii) The amino acid position corresponding to amino acid position 315 of the wild-type canine IgG, (xiii) The amino acid position corresponding to amino acid position 378 of the wild-type canine IgG, (xiv) The amino acid position corresponding to amino acid position 380 of the wild-type canine IgG, (xv) The amino acid position corresponding to amino acid position 428 of the wild-type canine IgG, (xvi) The amino acid position corresponding to amino acid position 430 of the wild-type canine IgG, (xvii) The amino acid position corresponding to amino acid position 433 of the wild-type canine IgG, (xviii) The amino acid position corresponding to amino acid position 434 of the wild-type canine IgG, and (xix) The polypeptide according to any one of the appendices 1 to 16, comprising at least one additional amino acid substitution at the amino acid position corresponding to amino acid position 435 of the wild-type canine IgG. (Note 18) The polypeptide described above, (i) The wild-type canine IgG contains Glu or Gln at the amino acid position corresponding to amino acid position 250, (ii) The wild-type canine IgG contains Asp or Glu at the amino acid position corresponding to amino acid position 251, (iii) containing Tyr or Met at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, (iv) The wild-type canine IgG contains Thr or Ser at the amino acid position corresponding to amino acid position 254, (v) The amino acid position corresponding to amino acid position 256 of the wild-type canine IgG contains Asp, Glu, or Phe, (vi) The wild-type canine IgG contains Asn or Asp at the amino acid position corresponding to amino acid position 285, (vii) The amino acid position corresponding to amino acid position 286 of the wild-type canine IgG contains Asp, Tyr, Phe, Leu, or Trp, (viii) The wild-type canine IgG contains Arg, Gln, or Ala at the amino acid position corresponding to amino acid position 307, (ix) The wild-type canine IgG contains Pro at the amino acid position corresponding to amino acid position 308, (x) containing Pro at the amino acid position corresponding to amino acid position 309 of the wild-type canine IgG, (xi) containing Val at the amino acid position corresponding to amino acid position 311 of the wild-type canine IgG, (xii) The wild-type canine IgG contains Asp at the amino acid position corresponding to amino acid position 315, (xiii) The wild-type canine IgG contains Val at the amino acid position corresponding to amino acid position 378, (xiv) The wild-type canine IgG contains Ala at the amino acid position corresponding to amino acid position 380, (xv) The wild-type canine IgG contains Leu at the amino acid position corresponding to amino acid position 428, (xvi) The wild-type canine IgG contains Ala or Lys at the amino acid position corresponding to amino acid position 430, (xvii) The wild-type canine IgG contains Lys at the amino acid position corresponding to amino acid position 433, (xviii) The amino acid position corresponding to amino acid position 434 of the wild-type canine IgG contains Trp, Tyr, Arg, His, Ser, Ala or Phe, and / or (xix) containing Tyr at the amino acid position corresponding to amino acid position 435 of the wild-type canine IgG, Polypeptides as described in Appendix 17. (Note 19) The position is selected from the following group: (i) The amino acid position corresponding to amino acid position 250 of the wild-type canine IgG, (ii) The amino acid position corresponding to amino acid position 251 of the wild-type canine IgG, (iii) The amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, (iv) The amino acid position corresponding to amino acid position 254 of the wild-type canine IgG, (v) The amino acid position corresponding to amino acid position 256 of the wild-type canine IgG, (vi) The amino acid position corresponding to amino acid position 285 of the wild-type canine IgG, (vii) The amino acid position corresponding to amino acid position 307 of the wild-type canine IgG, (viii) The amino acid position corresponding to amino acid position 308 of the wild-type canine IgG, (ix) The amino acid position corresponding to amino acid position 309 of the wild-type canine IgG, (x) The amino acid position corresponding to amino acid position 311 of the wild-type canine IgG, (xi) The amino acid position corresponding to amino acid position 315 of the wild-type canine IgG, (xii) The amino acid position corresponding to amino acid position 378 of the wild-type canine IgG, (xiii) The amino acid position corresponding to amino acid position 380 of the wild-type canine IgG, (xiv) The amino acid position corresponding to amino acid position 428 of the wild-type canine IgG, (xv) The amino acid position corresponding to amino acid position 430 of the wild-type canine IgG, (xvi) The amino acid position corresponding to amino acid position 433 of the wild-type canine IgG, (xvii) the amino acid position corresponding to amino acid position 434 of the wild-type canine IgG, and (xviii) the amino acid position corresponding to amino acid position 435 of the wild-type canine IgG A polypeptide as described in any one of the appendices 9 to 15, comprising at least one additional amino acid substitution. (Note 20) The polypeptide described above, (i) The wild-type canine IgG contains Glu or Gln at the amino acid position corresponding to amino acid position 250, (ii) The wild-type canine IgG contains Asp or Glu at the amino acid position corresponding to amino acid position 251, (iii) containing Tyr or Met at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, (iv) The wild-type canine IgG contains Thr or Ser at the amino acid position corresponding to amino acid position 254, (v) The amino acid position corresponding to amino acid position 256 of the wild-type canine IgG contains Asp, Glu, or Phe, (vi) The wild-type canine IgG contains Asn or Asp at the amino acid position corresponding to amino acid position 285, (vii) The wild-type canine IgG contains Arg, Gln, or Ala at the amino acid position corresponding to amino acid position 307, (viii) containing Pro at the amino acid position corresponding to amino acid position 308 of the wild-type canine IgG, (ix) The wild-type canine IgG contains Pro at the amino acid position corresponding to amino acid position 309, (x) The wild-type canine IgG contains Val at the amino acid position corresponding to amino acid position 311, (xi) containing Asp at the amino acid position corresponding to amino acid position 315 of the wild-type canine IgG, (xii) The wild-type canine IgG contains Val at the amino acid position corresponding to amino acid position 378, (xiii) The wild-type canine IgG contains Ala at the amino acid position corresponding to amino acid position 380, (xiv) The wild-type canine IgG contains Leu at the amino acid position corresponding to amino acid position 428, (xv) The amino acid position corresponding to amino acid position 430 of the wild-type canine IgG contains Ala or Lys, (xvi) containing Lys at the amino acid position corresponding to amino acid position 433 of the wild-type canine IgG, (xviii) The amino acid position corresponding to amino acid position 434 of the wild-type canine IgG contains Trp, Tyr, Arg, His, Ser, Ala or Phe, and / or (xviii) The polypeptide according to Appendix 19, comprising Tyr at the amino acid position corresponding to amino acid position 435 of the wild-type canine IgG. (Note 21) The position in which the aforementioned at least one additional amino acid substitution is selected from the group consisting of: (i) The amino acid position corresponding to amino acid position 250 of the wild-type canine IgG, (ii) The amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, (iii) The amino acid position corresponding to amino acid position 254 of the wild-type canine IgG, (iv) The amino acid position corresponding to amino acid position 256 of the wild-type canine IgG, (v) The amino acid position corresponding to amino acid position 285 of the wild-type canine IgG, (vi) the amino acid position corresponding to amino acid position 307 of the wild-type canine IgG, (vii) the amino acid position corresponding to amino acid position 309 of the wild-type canine IgG, (viii) The amino acid position corresponding to amino acid position 311 of the wild-type canine IgG, (ix) The amino acid position corresponding to amino acid position 315 of the wild-type canine IgG, (x) The amino acid position corresponding to amino acid position 433 of the wild-type canine IgG, and (xi) Amino acid position corresponding to amino acid position 434 of the wild-type canine IgG Polypeptides as described in Appendix 17 or Appendix 19. (Note 22) The polypeptide described above, (i) The wild-type canine IgG contains Glu or Gln at the amino acid position corresponding to amino acid position 250, (ii) The wild-type canine IgG contains Tyr or Met at the amino acid position corresponding to amino acid position 252, (iii) The wild-type canine IgG contains Thr or Ser at the amino acid position corresponding to amino acid position 254, (iv) The amino acid position corresponding to amino acid position 256 of the wild-type canine IgG contains Asp, Glu, or Phe, (v) The wild-type canine IgG contains Asn or Asp at the amino acid position corresponding to amino acid position 285, (vi) The wild-type canine IgG contains Arg, Gln, or Ala at the amino acid position corresponding to amino acid position 307, (vii) The wild-type canine IgG contains Pro at the amino acid position corresponding to amino acid position 309, (viii) The wild-type canine IgG contains Val at the amino acid position corresponding to amino acid position 311, (ix) The wild-type canine IgG contains Asp at the amino acid position corresponding to amino acid position 315, (x) containing Lys at the amino acid position corresponding to amino acid position 433 of the wild-type canine IgG, (xi) The polypeptide according to Appendix 21, wherein the amino acid position corresponding to amino acid position 434 of the wild-type canine IgG contains Trp, Tyr, Arg, His, Ser, Ala, or Phe. (Note 23) The position in which the aforementioned at least one additional amino acid substitution is selected from the group consisting of: (i) The amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, (ii) The amino acid position corresponding to amino acid position 254 of the wild-type canine IgG, (iii) The amino acid position corresponding to amino acid position 256 of the wild-type canine IgG, and (iv) The amino acid position corresponding to amino acid position 434 of the wild-type canine IgG Polypeptides as described in Appendix 17 or Appendix 19. (Note 24) The polypeptide described above, (i) The wild-type canine IgG contains Tyr or Met at the amino acid position corresponding to amino acid position 252, (ii) The wild-type canine IgG contains Thr or Ser at the amino acid position corresponding to amino acid position 254, (iii) containing Asp, Glu, or Phe at the amino acid position corresponding to amino acid position 256 of the wild-type canine IgG, and / or (iv) The polypeptide described in Appendix 23, which contains Trp, Tyr, Arg, His, Ser, Ala, or Phe at the amino acid position corresponding to amino acid position 434 of wild-type canine IgG. (Note 25) The polypeptide described above, (i) containing Tyr at the amino acid position corresponding to amino acid position 252 of the wild-type canine IgG, (ii) The wild-type canine IgG contains Thr at the amino acid position corresponding to amino acid position 254, (iii) containing Glu at the amino acid position corresponding to amino acid position 256 of the wild-type canine IgG, and / or (iv) The polypeptide described in Appendix 24, wherein the amino acid position corresponding to amino acid position 434 of the wild-type canine IgG contains Trp, Tyr, Arg, or His. (Note 26) A polypeptide comprising a canine IgG Fc region variant or its canine FcRn binding region, comprising two or more positions selected from the group consisting of: (i) The position corresponding to amino acid position 286 of wild-type canine IgG; (ii) The position corresponding to amino acid position 312 of wild-type canine IgG; (iii) The position corresponding to amino acid position 426 of wild-type canine IgG; (iv) the position corresponding to amino acid position 434 of wild-type canine IgG; and (v) The polypeptide comprising an amino acid substitution at the position corresponding to amino acid position 436 of wild-type canine IgG, wherein the amino acid position is determined based on EU numbering, and the polypeptide has increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG. (Note 27) The polypeptide according to Appendix 26, wherein the amino acid substitution at the position corresponding to amino acid position 286 of wild-type canine IgG is selected from the group consisting of T286L, T286Y, and any of the aforementioned conservative amino acid substitutions. (Note 28) The polypeptide according to Appendix 26, wherein the amino acid substitution at the position corresponding to amino acid position 312 of wild-type canine IgG is D312P or a conserved amino acid substitution thereof. (Note 29) The polypeptide according to Appendix 26, wherein the amino acid substitution at the position corresponding to amino acid position 426 of wild-type canine IgG is selected from the group consisting of A426Y, A426H, and any of the aforementioned conservative amino acid substitutions. (Note 30) The polypeptide according to Appendix 26, wherein the amino acid substitution at the position corresponding to amino acid position 434 of wild-type canine IgG is N434R or a conserved amino acid substitution thereof. (Note 31) The polypeptide according to Appendix 26, wherein the amino acid substitution at the position corresponding to amino acid position 436 of wild-type canine IgG is Y436H or a conserved amino acid substitution thereof. (Note 32) The polypeptide according to any one of the appendices 26 to 31, wherein the polypeptide includes an amino acid substitution at the position corresponding to amino acid position 426 of wild-type canine IgG. (Note 33) The polypeptide has two or more positions selected from the group consisting of the following: (i) Positions corresponding to amino acid positions 426 and 286 of wild-type canine IgG; (ii) Positions corresponding to amino acid positions 426 and 312 of wild-type canine IgG; (iii) Positions corresponding to amino acid positions 426 and 434 of wild-type canine IgG; (iv) The positions corresponding to amino acid positions 426 and 436 of wild-type canine IgG; and (v) The polypeptide described in Appendix 32, comprising amino acid substitutions at positions corresponding to amino acid positions 286, 426, and 436 of wild-type canine IgG. (Note 34) The polypeptide is subjected to amino acid substitutions selected from the following group: (i) A426Y and T286L; (ii) A426Y and D312P; (iii) A426Y and Y436H; (iv) A426H and T286L; (v) A426H and T286Y; (vi) A426H and D312P; and (vii) T286L, A426Y, and Y436H Polypeptides as described in Appendix 32, including the polypeptides described in Appendix 32. (Note 35) The two or more amino acid substitutions described above (i) A combination of A426Y and one or more of T286L, D312P, N434R, and Y436H; (ii) combinations of A426H with one or more of T286L, T286Y, D312P, N434R and Y436H; and (iii) A polypeptide according to any one of the appendices 26 to 31, selected from the group consisting of combinations of N434R and one or more of T286L, T286Y, D312P, and Y436H. (Note 36) The polypeptide according to any one of the appendices 1 to 35, wherein the wild-type canine IgG is canine IgGA containing an Fc domain having an amino acid sequence at least 80% identical to SEQ ID NO: 9, canine IgGB containing an Fc domain having an amino acid sequence at least 80% identical to SEQ ID NO: 10, canine IgGC containing an Fc domain having an amino acid sequence at least 80% identical to SEQ ID NO: 11, or canine IgGD containing an Fc domain having an amino acid sequence at least 80% identical to SEQ ID NO: 12. (Note 37) The polypeptide according to any one of the appendices 1 to 36, wherein the wild-type canine IgG is canine IgGA, and the Fc region variant of the canine IgG or its canine FcRn binding region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 9. (Note 38) The polypeptide according to any one of the appendices 1 to 36, wherein the wild-type canine IgG is canine IgGB, and the Fc region variant of the canine IgG or its canine FcRn binding region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 10. (Note 39) The polypeptide according to any one of the appendices 1 to 36, wherein the wild-type canine IgG is canine IgGC, and the Fc region variant of the canine IgG or its canine FcRn binding region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 11. (Note 40) The polypeptide according to any one of the appendices 1 to 36, wherein the wild-type canine IgG is canine IgGD, and the Fc region variant of the canine IgG or its canine FcRn binding region comprises an amino acid sequence that is at least 80% identical to SEQ ID NO: 12. (Note 41) A polypeptide as described in any one of the appendices 1 to 40, further comprising a binding domain. (Note 42) The aforementioned binding domain, (i) Six complementarity-determining regions (CDRs) of immunoglobulin molecules; (ii) Ligand-binding domain of canine receptor protein, (iii) nanobody, or (iv) A polypeptide as described in Appendix 41, comprising the extracellular domain of a canine receptor protein. (Note 43) The polypeptide according to Appendix 41 or Appendix 42, wherein the binding domain specifically binds to an antigen selected from the group consisting of NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-33, CD3, CD20, CD47, CD52, and the complement system complex. (Note 44) A polypeptide as described in any one of the appendices 1 to 43, further comprising a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, GLP-1 receptor agonists, and thrombopoietin-binding peptides. (Note 45) The polypeptide described in any one of the appendices 1 to 44, wherein the polypeptide binds to canine FcRn at a higher level at an acidic pH than at a neutral pH in the binding assay. (Note 46) The polypeptide described in Appendix 45, wherein the polypeptide binds to canine FcRn at a higher level at pH 5.5 than at pH 7.4 in the binding assay. (Note 47) The polypeptide described in Appendix 46, wherein the polypeptide binds to canine FcRn at a higher level at pH 6.0 than at pH 7.4 in the binding assay. (Note 48) A pharmaceutical composition comprising (i) a polypeptide as described in any one of Appendix 1 to 47, and (ii) a pharmaceutically acceptable excipient. (Note 49) One or more nucleic acids encoding a polypeptide as described in any one of the appendices 1 to 47. (Note 50) One or more expression vectors comprising one or more nucleic acids as described in Appendix 49. (Note 51) A host cell containing one or more nucleic acids as described in Appendix 49 or one or more expression vectors as described in Appendix 50. (Note 52) A method for producing polypeptides, (a) To provide one or more nucleic acids as described in Appendix 49; (b) Expressing one or more nucleic acids in a host cell culture to produce the polypeptide; and The method comprising recovering the polypeptide produced in (c)(b) from the host cell culture. (Note 53) The method described in Appendix 52, further comprising formulating the polypeptide as a pharmaceutical preparation. (Note 54) A method for treating a disease or disorder in a dog in need thereof, comprising administering to the dog an effective amount of a composition containing the pharmaceutical composition described in Appendix 48. (Note 55) A method for preventing a disease or disorder in a dog in need thereof, comprising administering to the dog an effective amount of a composition containing the pharmaceutical composition described in Appendix 48. (Note 56) The method according to Appendix 54 or Appendix 55, wherein the disease or disorder of the dog is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disease, gastrointestinal disease, cardiovascular disease, kidney disease, reproductive function disorder, infection, or cancer. (Note 57) The method according to Appendix 54 or Appendix 55, wherein the disease or disorder of the dog is atopic dermatitis, allergic dermatitis, osteoarthralgia, arthritis, anemia, or obesity. (Note 58) A pharmaceutical composition as described in Appendix 48, for use in a manner that treats a disease or disorder in a dog in need thereof. (Note 59) A pharmaceutical composition as described in Appendix 48, for use in a manner that prevents a disease or disorder in a dog in need thereof. (Note 60) A pharmaceutical composition for use as described in Appendix 58 or Appendix 59, wherein the disease or disorder of the dog is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disorder, gastrointestinal disorder, cardiovascular disease, kidney disease, reproductive dysfunction, infection, or cancer. (Note 61) A pharmaceutical composition for use as described in Appendix 58 or Appendix 59, wherein the disease or disorder of the dog is atopic dermatitis, allergic dermatitis, osteoarthralgia, arthritis, anemia, or obesity. (Note 62) Use of polypeptides described in any one of Annexes 1 to 47 in the manufacture of a drug for treating a disease or disorder in dogs in need thereof. (Note 63) Use of polypeptides described in any one of Annexes 1 to 47 in the manufacture of a drug for preventing disease or disability in dogs in need thereof. (Note 64) The use described in Appendix 62 or Appendix 63, wherein the disease or disorder of the dog is an allergic disease, chronic pain, acute pain, inflammatory disease, autoimmune disease, endocrine disorder, gastrointestinal disorder, cardiovascular disease, kidney disease, reproductive dysfunction, infection, or cancer. (Note 65) The use described in Appendix 62 or 63, wherein the disease or disorder of the dog is atopic dermatitis, allergic dermatitis, osteoarthralgia, arthritis, anemia, or obesity.

[0244] Other Embodiments The present invention has been described in detail, but the above description is illustrative of the scope of the invention and is not intended to limit it. The present invention is defined by the appended claims. Other aspects, advantages, and modifications are within the scope of the following claims.

Claims

1. A polypeptide comprising a canine IgG Fc region variant or a canine FcRn binding region thereof, wherein the polypeptide comprises at least one amino acid substitution selected from the group consisting of (i) an amino acid substitution comprising Tyr, Phe, Leu, or Trp at the position corresponding to amino acid position 286 of wild-type canine IgG; (ii) an amino acid substitution comprising Pro at the position corresponding to amino acid position 312 of wild-type canine IgG; (iii) an amino acid substitution comprising Tyr, His, or Phe at the position corresponding to amino acid position 426 of wild-type canine IgG; and (iv) an amino acid substitution comprising His at the position corresponding to amino acid position 436 of wild-type canine IgG. Here, the amino acid positions are based on EU numbering, and the polypeptide has increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG. The polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 12.

2. The polypeptide described above, (i) an amino acid substitution containing Glu or Glu at the position corresponding to amino acid position 250 of the wild-type canine IgG, (ii) an amino acid substitution containing Asp or Glu at the position corresponding to amino acid position 251 of the wild-type canine IgG, (iii) an amino acid substitution containing Tyr or Met at the position corresponding to amino acid position 252 of the wild-type canine IgG, (iv) an amino acid substitution containing Thr or Ser at the position corresponding to amino acid position 254 of the wild-type canine IgG, (v) an amino acid substitution at amino acid position 256 of the wild-type canine IgG (vi) an amino acid substitution containing Asp, Glu, or Phe at the corresponding position, (vii) an amino acid substitution containing Asn or Asp at the position corresponding to amino acid position 285 of the wild-type canine IgG, (vii) an amino acid substitution containing Asp, Tyr, Phe, Leu, or Trp at the position corresponding to amino acid position 286 of the wild-type canine IgG, (viiii) an amino acid substitution containing Arg, Gln, or Ala at the position corresponding to amino acid position 307 of the wild-type canine IgG, (ix) corresponding to amino acid position 308 of the wild-type canine IgG (x) an amino acid substitution containing Pro at the position, (x) an amino acid substitution containing Pro at the position corresponding to amino acid position 309 of the wild-type canine IgG, (xi) an amino acid substitution containing Val at the position corresponding to amino acid position 311 of the wild-type canine IgG, (xi) an amino acid substitution containing Asp at the position corresponding to amino acid position 315 of the wild-type canine IgG, (xi) an amino acid substitution containing Val at the position corresponding to amino acid position 378 of the wild-type canine IgG, (xi) an amino acid substitution containing Val at the position corresponding to amino acid position 380 of the wild-type canine IgG (xv) an amino acid substitution containing Ala at the position corresponding to amino acid position 428 of the wild-type canine IgG, (xvi) an amino acid substitution containing Ala or Lys at the position corresponding to amino acid position 430 of the wild-type canine IgG, (xvii) an amino acid substitution containing Lys at the position corresponding to amino acid position 433 of the wild-type canine IgG, (xviiii) an amino acid substitution containing Trp, Tyr, Arg, His, Ser, Ala or Phe at the position corresponding to amino acid position 434 of the wild-type canine IgG,and / or (xix) the polypeptide according to claim 1, further comprising at least one amino acid substitution selected from the group consisting of amino acid substitutions containing Tyr at the position corresponding to amino acid position 435 of the wild-type canine IgG.

3. A polypeptide comprising a canine IgG Fc region variant or a canine FcRn binding region thereof, wherein the polypeptide comprises two or more amino acid substitutions selected from the group consisting of (i) an amino acid substitution containing Tyr, Phe, Leu, or Trp at the position corresponding to amino acid position 286 of wild-type canine IgG; (ii) an amino acid substitution containing Pro at the position corresponding to amino acid position 312 of wild-type canine IgG; (iii) an amino acid substitution containing Tyr, His, or Phe at the position corresponding to amino acid position 426 of wild-type canine IgG; (iv) an amino acid substitution containing Arg at the position corresponding to amino acid position 434 of wild-type canine IgG; and (v) an amino acid substitution containing His at the position corresponding to amino acid position 436 of wild-type canine IgG. Here, the amino acid positions are based on EU numbering, and the polypeptide has increased binding affinity to canine FcRn compared to the Fc domain of wild-type canine IgG. The polypeptide comprises an amino acid sequence that is at least 90% identical to an amino acid sequence selected from the group consisting of SEQ ID NOs: 9 to 12.

4. The polypeptide has amino acid substitutions selected from the group consisting of the following: (i) A426Y and T286Y; (ii) A426Y and T286F; (iii) A426Y and T286L; (iv) A426Y and T286W; (v) A426Y and D312P; (vi) A426Y and Y436H; (vii) A426H and T286Y; (viiii) A426H and T286F; (ix) A426H and T286L; (x) A426H and T286W; (xi) A426H and D312P; (xi) A426F and T286Y; (xiii) A426F and T286F; (xiv) A426F and T286L; (xv) A426F and T286W; and (xvi) T286L, A426Y, and Y436H The polypeptide according to claim 3, comprising:

5. The polypeptide according to any one of claims 1 to 4, wherein the wild-type canine IgG is canine IgGA comprising an Fc domain having an amino acid sequence at least 90% identical to SEQ ID NO: 9, canine IgGB comprising an Fc domain having an amino acid sequence at least 90% identical to SEQ ID NO: 10, canine IgGC comprising an Fc domain having an amino acid sequence at least 90% identical to SEQ ID NO: 11, or canine IgGD comprising an Fc domain having an amino acid sequence at least 90% identical to SEQ ID NO:

12.

6. It further includes a binding domain, The aforementioned binding domain is optionally selected, (i) Six complementarity-determining regions (CDRs) of immunoglobulin molecules, (ii) Ligand-binding domain of canine receptor protein, (iii) Nanobody, or (iv) Containing the extracellular domain of the canine receptor protein, The binding domain may optionally bind specifically to an antigen selected from the group consisting of NGF, TrKA, ADAMTS, IL-1, IL-2, IL-4, IL-4R, angiotensin type 1 (AT1) receptor, angiotensin type 2 (AT2) receptor, IL-5, IL-12, IL-13, IL-31, IL-33, CD3, CD20, CD47, CD52, and the complement system complex. The polypeptide according to any one of claims 1 to 5.

7. The polypeptide according to any one of claims 1 to 6, further comprising a protein selected from the group consisting of EPO, CTLA4, LFA3, VEGFR1 / VEGFR3, IL-1R, IL-4R, GLP-1 receptor agonists, and thrombopoietin-binding peptides.

8. The polypeptide according to any one of claims 1 to 7, wherein the polypeptide binds to canine FcRn at a higher level at an acidic pH than at a neutral pH in the binding assay.

9. The polypeptide according to claim 8, wherein the polypeptide binds to canine FcRn at a higher level at pH 6.0 than at pH 7.4 in the binding assay.

10. (i) a polypeptide according to any one of claims 1 to 9, and (ii) a pharmaceutically acceptable excipient.

11. One or more nucleic acids encoding a polypeptide according to any one of claims 1 to 9.

12. One or more expression vectors comprising one or more nucleic acids according to claim 11.

13. A host cell comprising one or more nucleic acids according to claim 11 or one or more expression vectors according to claim 12.

14. A method for producing polypeptides, (a) To provide one or more nucleic acids as described in claim 11; (b) Expressing one or more nucleic acids in a host cell culture to produce the polypeptide; and (c) Recovering the polypeptide produced in (b) from the host cell culture. The method further optionally comprises formulating the polypeptide as a pharmaceutical product.

15. A composition comprising the pharmaceutical composition according to claim 10, used for the treatment or prevention of a disease or disorder in a dog that requires treatment or prevention, wherein the disease or disorder in the dog is (a) allergic diseases, chronic pain, acute pain, inflammatory diseases, autoimmune diseases, endocrine disorders, gastrointestinal diseases, cardiovascular diseases, kidney diseases, reproductive disorders, infections or cancer, (b) A composition for atopic dermatitis, allergic dermatitis, osteoarthralgia, arthritis, anemia, or obesity.

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